Anti-cd40 antibody mutants

ABSTRACT

A mutant of a potentially therapeutic anti-CD40 antibody is provided which mutant has reduced ADCC and CDC activities designed to be optimized as a pharmaceutical agent. A mutant of an agonistic anti-CD40 antibody, comprising mutation and/or substitution of at least one amino acid in the constant region to reduce the ADCC and/or CDC activities therein, and a mutant of an antagonistic anti-CD40 antibody, comprising at least one mutation or substitution in the constant region to reduce the ADCC and/or CDC activities therein, both mutants having at least a hinge region derived from a human IgG2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/584,345, filed Feb. 26, 2007, which is a U.S. National Phase ofInternational Application PCT/JP2004/019750, filed Dec. 24, 2004, whichwas published on Jul. 14, 2005, as WO 2005/063981, which claims thebenefit of JP Application No. 2003-431408, filed Dec. 25, 2003, all ofwhich are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an anti-CD40 antibody which recognizesCD40 which is a type of cell membrane molecules associated withimmunity. Further, the present invention relates to an antibody with amutation in the constant region of the human antibody or with a subclasshaving its portion substituted in order to decrease an ADCC and/or CDCactivity, while keeping an agonistic or antagonistic activity.

BACKGROUND ART 1. CD40

CD40 is an antigen having a molecular weight of 50 kDa which is presenton the surface of cell membrane, and expressed in B cells, dendriticcells (DCs), some types of cancer cells, and thymic epithelial cells.CD40 is known to play an important role in proliferation anddifferentiation of B cells and DCs. CD40 was identified as an antigenexpressed on the surface of human B cells (E. A. Clark et al., Proc.Natl. Acad. Sci. USA 83: 4494, 1986; and I. Stamenkovic et al., EMBO J.8: 1403, 1989) and has been considered as a member of the TNF receptorfamily which includes low-affinity NGF receptors, TNF receptors, CD27,OX40 and CD30. Ligands (CD40Ls) to human and murine CD40s have beenrecently cloned and found to be membrane proteins type II and expressedin activated CD4+T cells. CD40L has been also found to introduce strongsignals for activation into human or murine B cells.

In dendritic cells, CD40 has been observed to be more highly expressedthan in B cells, and it has become clear that CD40 plays an importantrole in dendritic cells. Binding of CD40 to CD40L activates antigenpresenting cells (APCs), that is, expresses costimulator molecules suchas CD80 (B7-1) and CD86 (B7-2) or enhances production of IL-2 (Caux, C.,et al.: Activation of human dendritic cells through CD40 cross-linking.J. Exp. Med., 180: 1263, 1994; and Shu, U., et al.: Activated T cellsinduce interleukin-12 production by monocyte via CD40-CD40 ligandinteraction. Eur. J. Immunol., 25: 1125, 1995). Dendritic cells have astrong antigen-presenting capacity and a strong capacity to activatehelper T (Th) cells. Dendritic cells are also believed to controldifferentiation of naive Th cells into Th1 or Th2 cells. When peripheralblood monocytes, which are myeloid dendritic cells, are cultured in thepresence of GM-CSF and IL-4, and matured by CD40L, the resulting matureddendritic cells (DC1) can produce IL-12 in vitro, and stimulate andactivate allogeneic naive Th cells to induce IFNγ-producing T cells(i.e., to promote their differentiation into Th1). This action isinhibited by anti-IL-12 antibody and hence may be effected via IL-12. Onthe other hand, when plasmacytoid T cells, which are present in lymphoidT regions and peripheral blood, are cultured in the presence of IL-3 andCD40 ligand, the resulting lymphoid dendritic cells (DC2) are shown tobe unable to produce IL-12, and stimulate and activate allogeneic naiveTh cells to induce IL-4-producing T cells, which indicates promotion oftheir differentiation into Th2. It is believed that Th1 cells areinvolved in activation of cellular immunity, while Th2 cells areassociated with enhancement of humoral immunity as well as restrictionof cellular immunity. When cytotoxic T cells (CTL) are activated withthe help of Th1 cells, they may eliminate pathogens (a number of typesof virus, listeria, tuberculosis bacteria, toxoplasma protozoa, etc.)growing in the cytoplasm and tumor cells.

Monoclonal anti-CD40 antibodies, which recognize CD40 expressed on themembrane surface, have been demonstrated to have different biologicalactivities to B cells. Monoclonal anti-CD40 antibodies are generallyclassified into agonistic or antagonistic antibodies against theinteraction between CD40 and CD40L.

2. Agonistic Antibodies

Agonistic antibodies are known to activate B cells. For instance, theanti-CD40 antibodies are reported to induce cell adhesion (Barrett etal., J. Immunol. 146: 1722, 1991; and Gordon et al., J. Immunol. 140:1425, 1998), increase cell size (Gordon et al., J. Immunol. 140: 1425,1998; and Valle et al., Eur. J. Immunol. 19: 1463, 1989), induce celldivision of B cells activated only by an anti-IgM antibody, anti-CD20antibody or phorbol ester (Clark and Ledbetter, Proc. Natl. Acad. Sci.USA 83: 4494, 1986; Gordon et al., LEUCOCYTE TYPING III. A. J. McMichealed. Oxford University Press. Oxford. p. 426; and Paulie et al., J.Immunol. 142: 590, 1989), induce cell division of B cells in thepresence of IL4 (Valle et al., Eur. J. Immunol. 19: 1463, 1989; andGordon et al., Eur. J. Immunol. 17: 1535, 1987), induce expression ofIgE by cultured cells stimulated with IL-4 and deprived of T cells(Jabara et al., J. Exp. Med. 172: 1861, 1990; and Gascan et al., J.Immunol. 147: 8, 1991), induce expression of IgG and IgM by thosecultured cells (Gascan et al., J. Immunol. 147: 8, 1991), secretesoluble CD23/FceRII from cells via IL-4 (Gordon and Guy, Immunol. Today8: 39, 1987; and Cairns et al., Eur. J. Immunol. 18: 349, 1988), enhanceexpression of soluble CD23/FceRII on the cells via IL4 (Challa, A.,Allergy, 54: 576, 1999), and promote IL-6 production (Clark and Shu, J.Immunol. 145: 1400, 1990). Furthermore, it is reported that addition ofIL-4 and an anti-CD40 antibody to human primary culture B cells in thepresence of CDw32+adhesive cells led to establishment of cloned B cellsderived therefrom (Bancherauet et al., Science 241: 70, 1991), andapoptosis of germinal center cells was inhibited through CD40irrespective of whether its antigen receptor was active or inactive (Liuet al., Nature 342: 929, 1989). As described above, CD40 has beenidentified as antigen expressed on the surface of human B cells, andconsequently, most of the isolated antibodies have been evaluated, as anindex, mainly using their induction potency for proliferation and/ordifferentiation of human B cells, or their induction activity for celldeath of cancer cells (Katira, A. et al., LEUKOCYTE TYPING V. S. F.Schlossossman, et. al. eds. p. 547. Oxford University Press. Oxford; W.C. Flansow et. al., LEUKOCYTE TYPING V. S. F. Schlossossman, et. al.eds. p. 555. Oxford University Press. Oxford; and J. D. Pound et. al.,International Immunology, 11: 11, 1999).

The anti-CD40 antibody has been demonstrated to mature DC (Z. H. Zhouet. al., Hybridoma, 18: 471, 1999). Furthermore, the role of CD4 T cellsin priming antigen-specific CD8 T cells was reported to be in activationof DC via CD40-CD40L signaling, and the anti-CD40 monoclonal antibody(mAb) has been found to be able to replace CD40 helper T cells inactivation of dendritic cells (DC) (Shoenberger, S. P., et. al.: T-cellhelp for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions.Nature, 480, 1998). Also, administration of an anti-CD40 antibody inmice has been found to be able to protect the animal body fromCD40-expressing tumor cells as well as CD40-non-expressing tumor cells(French, R. R., et. al.: CD40 antibody evokes a cytotoxic T-cellresponse that eradicates lymphoma and bypasses T-cell help. NatureMedicine, 5, 1999).

Agonistic anti-CD40 antibodies are expected to be effective fortreatment of infectious diseases, due to bacteria, virus, etc., cancerand others, based on their functions described above. Anti-CD40antibodies with superior agonistic activities are described in WO02/088186. The representative examples of those agonistic antibodies areKM341-1-19 and 2105 antibodies. The hybridoma KM341-1-19 producing theKM341-1-19 antibody and the hybridoma 2105 producing the 2105 antibodywere submitted on 27, Sep. 2001 and 17, Apr. 2002, respectively, forinternational deposit under the Budapest Treaty, to International PatentOrganisms Depositary, National Institute of Advanced Industrial Scienceand Technology (central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan).Their accession numbers are FERM BP-7759 (KM341-1-19) and FERM BP-8024(2105).

3. Antagonistic Antibodies

Taking it in consideration, on the other hand, that CD40 plays animportant role in immunologic responses, as aforementioned, it isexpected that inhibition of binding of CD40 to its ligands would lead todevelopment of therapeutic agents for immune suppression in organtransplantation and autoimmune diseases. Sawada, Hase and others havereported that the peripheral blood of patients suffering from Crohn'sdisease has a higher percentage of monocytes highly expressing CD40.However, such antibodies have not been well known yet as inhibit bindingof CD40 to its ligands. Those inhibitory antibodies would be useful infunctional analysis of CD40 and treatment of diseases requiringactivation of CD40. Inhibitory antibodies to CD40 ligands are alsosuggested to be effective against diseases involving binding of CD40 tothe CD40 ligands. However, CD40L was reported to be expressed inactivated platelets (V. Henn et al., Nature 391: 591, 1998), and if ananti-CD40L antibody is used as a therapeutic agent, thrombus formationmay occur reportedly (T. Kawai et al., Nat. Med. 6: 114, 2000). Fromthis point of view, antibodies to CD40 are expected to be safer ratherthan anti-CD40L antibodies as therapeutic antibody agent to inhibitbinding of CD40 to its ligands. Anti-CD40 antibodies would be requiredto inhibit binding of CD40L to CD40 and still not activate CD40 inthemselves.

Such antagonistic anti-CD40 antibodies may be used for treatment ofautoimmune diseases and suppression of immunologic rejections intransplantation of organs, bone marrow, etc., in view of their functionsdescribed above. Anti-CD40 antibodies with superior antagonisticactivities are described in WO 02/088186. The representative example ofthose antagonistic antibodies is 4D11 antibody. The hybridoma 4D11producing the 4D11 antibody was submitted on 27, Sep. 2001 forinternational deposit under the Budapest Treaty, to International PatentOrganisms Depositary, National Institute of Advanced Industrial Scienceand Technology (central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan). Theaccession number is FERM BP-7758.

-   Patent Document 1 WO 02/088186

DISCLOSURE OF THE INVENTION

The object of the present invention is to create mutants from thepotentially therapeutic anti-CD40 antibodies disclosed in WO 02/088186,which mutants are designed optimally as pharmaceutical agent.

As a result of extensive and intensive research, the present inventorshave successfully created novel mutants of the agonistic or antagonisticantibodies, which mutants may have a higher therapeutic effect againstdiseases than known anti-CD40 antibodies, and completed the presentinvention based thereon. The basic idea on modification of the anti-CD40antibodies according to the present invention will be described indetail below.

The present specification shall encompass the description in thespecification and/or drawings of JP Patent Publication (Kokai) No.2003-431408 which is the basis for the priority of the presentapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1 shows binding site peptides (SEQ ID NOs: 49-88) prepared basedon the CD40 sequence, to which the anti-CD40 agonistic antibodies bind;

FIG. 1A-2 shows binding site peptides (SEQ ID NOs: 89-130) preparedbased on the CD40 sequence, to which the anti-CD40 agonistic antibodiesbind (a continuation to FIG. 1A-1);

FIG. 1B-1 shows binding site peptides (SEQ ID NOs: 49-88) prepared basedon the CD40 sequence, to which the anti-CD40 antagonistic antibodiesbind;

FIG. 1B-2 shows binding site peptides (SEQ ID NOs: 89-130) preparedbased on the CD40 sequence, to which the anti-CD40 antagonisticantibodies bind (a continuation to FIG. 1B-1);

FIG. 2A illustrates binding of the anti-CD40 antibodies to the CD40mutant;

FIG. 2B illustrates binding of the anti-CD40 antibodies to the CD40mutant;

FIG. 2C illustrates binding of the anti-CD40 antibodies to the CD40mutant;

FIG. 3A shows diagrams indicating that the KM341-1-19 antibody having aP331S mutation is as active as the original KM341-1-19 antibody withrespect to binding to Ramos cells;

FIG. 3B shows diagrams indicating that the KM341-1-19 antibody having aP331S mutation is as active as the original KM341-1-19 antibody withrespect to enhancement of CD95 expression of Ramos cells;

FIG. 4A shows a diagram indicating that the KM341-1-19 antibody having aP331S mutation has a lower CDC activity via the rabbit complement;

FIG. 4B shows a diagram indicating that the G2/4 antibody has a lowercomplement activity when the human complement is used;

FIG. 5A-1 shows diagrams indicating that conversion of the subclass ofthe 2105 antibody from IgG2 into different subclasses has no effect onits binding to Ramos cells;

FIG. 5A-2 shows diagrams indicating that conversion of the subclass ofthe KM341-1-19 antibody from IgG2 into different subclasses has noeffect on its binding to Ramos cells;

FIG. 5B-1 shows diagrams indicating that conversion of the subclass ofthe 2105 antibody from IgG2 into different subclasses lowers an activityto enhance CD95 expression of Ramos cells;

FIG. 5B-2 shows diagrams indicating that conversion of the subclass ofthe KM341-1-19 antibody from IgG2 into different subclasses lowers anactivity to enhance CD95 expression of Ramos cells;

FIG. 6A-1 shows diagrams indicating that the binding capacity of theKM341-1-19 antibodies to Ramos cells is independent of the varyingstructure of the hinge region;

FIG. 6A-2 shows diagrams indicating that the binding capacity of the2105 antibodies to Ramos cells is independent of the varying structureof the hinge region;

FIG. 6B-1 shows diagrams indicating that the upper and middle hinges ofthe hinge region are important for the activity of the KM341-1-19antibodies to enhance CD95 expression of Ramos cells;

FIG. 6B-2 shows diagrams indicating that the upper and middle hinges ofthe hinge region are important for the activity of the 2105 antibodiesto enhance CD95 expression of Ramos cells;

FIG. 7A shows diagrams indicating that conversion of the subclass of theF72 antibody to IgG2 has no effect on its binding to Ramos cells;

FIG. 7B shows diagrams indicating that conversion of the subclass of theF72 antibody to IgG2 raises an activity to enhance CD95 expression ofRamos cells;

FIG. 8A shows diagrams indicating that conversion of the subclass of the4D11 antibody from IgG1 to IgG4 has no effect on its binding to Ramoscells;

FIG. 8B shows diagrams indicating that conversion of the subclass of the4D11 antibody from IgG1 to IgG4 inhibits enhancement by the CD40Ligandof CD95 expression of Ramos cells, at the same extent as otherwise;

FIG. 9 shows a diagram indicating that conversion of the subclass of the4D11 antibody from IgG1 to IgG4 or IgG4PE lowers the ADCC activity;

FIG. 10 shows a diagram indicating that conversion of the subclass ofthe 4D11 antibody from IgG1 to IgG4P lowers the CDC activity;

FIG. 11 illustrates a variation in number of B cells in the blood(B220-positive cells among the peripheral blood lymphocytes) over timeafter 4D11G1, 4D11G4P or 4D11G4PE was administered into humanCD40-transgenic mice;

FIG. 12A illustrates a higher expression of CD23 of splenic B cells(CD23-positive cells among the splenic B cells) after each anti-CD40antibody was administered into human CD40-transgenic mice;

FIG. 12B illustrates a higher expression of CD86 of splenic B cells(CD86-positive cells among the splenic B cells) after each anti-CD40antibody was administered into human CD40-transgenic mice;

FIG. 12C illustrates a higher expression of CD95 of splenic B cells(CD95-positive cells among the splenic B cells) after each anti-CD40antibody was administered into human CD40-transgenic mice;

FIG. 13A illustrates the suppressive activity of the antigen-specificantibody (IgG1) production by 4D11 and 281-1-10 in human CD40-transgenicmice;

FIG. 13B illustrates the suppressive activity of the antigen-specificantibody (IgM) production by 4D11 and 281-1-10 in human CD40-transgenicmice;

FIG. 14A illustrates the numbers of B cells in the blood (B220-positivecells among the peripheral blood lymphocytes) during the suppressionassay of the antigen-specific antibody producing activity;

FIG. 14B illustrates the numbers of splenic B cells (B220-positive cellsamong the splenic lymphocytes) during the suppression assay of theantigen-specific antibody producing activity;

FIG. 15 illustrates a variation in number of B cells in the blood(B220-positive cells among the peripheral blood lymphocytes) over timeafter 4D11G4P or 4D11G4PE was administered at a dose of 30 mg/kg intocynomolgus monkeys;

FIG. 16 illustrates blood IL-12 levels during the assay shown in FIG.15;

FIG. 17 shows the suppressive effect of 4D11G4PE on the simian DTH(delayed-type hypersensitivity in male cynomolgus monkeys);

FIG. 18 shows the titers of the anti tetanus toxin IgG during the assaywith the results shown in FIG. 17;

FIG. 19 shows the titers of the anti tetanus toxin IgM during the assaywith the results shown in FIG. 17;

FIG. 20A illustrates the respective influences of 4D11G4PE and 5C8(anti-CD40Ligand antibody) on platelet aggregation;

FIG. 20B shows the respective influences of 4D11G4PE and 5C8(anti-CD40Ligand antibody) on platelet aggregation;

FIG. 21 illustrates a variation in oligomer content of 4D11G4P,4D11G4PE, 4D11G2Ser or 4D11G4/2/4 over time after it was incubated at pH2.7 and 37° C.;

FIG. 22 illustrates suppression of rejection of skin grafts by theanti-CD40 antagonistic antibody;

FIG. 23 illustrates the volume change of the tumor over time from cellimplantation, in a case where 341G2Ser was administered to tumor bearingmice with Ramos cells implanted therein;

FIG. 24 illustrates the volume change of the tumor over time from cellimplantation, in a case where 341G2Ser was administered to tumor bearingmice with T24 cells implanted therein;

FIG. 25 illustrates the volume change of the tumor over time from cellimplantation, in a case where 341G2Ser was administered to tumor bearingmice with Hs 766T cells implanted therein; and

FIG. 26 illustrates the volume change of the tumor over time from cellimplantation, in a case where 341G2Ser was administered to tumor bearingmice with Capan-2 cells implanted therein.

BEST MODE FOR CARRYING OUT THE INVENTION 1. Modification of AgonisticAntibodies

Antibodies are essentially molecules that function to protect livingbodies against foreign bodies, such as microorganisms and viruses, andcancer, and hence they can kill and eliminate such cells binding tothemselves. The lethal activity is composed of two different activities,called Antibody-Dependent Cellular Cytotoxicity (abbreviated as ADCChereinafter) and Complement-Dependent Cytotoxicity (abbreviated as CDChereinafter).

ADCC refers to a type of cytotoxicity induced by activation ofmacrophages, NK cells, neutrophil cells, etc., which recognize targetcells by binding to the constant region of the antibody via Fc receptorsexpressed on their surface. In contrast, CDC refers to a type ofcytotoxicity induced by activation of a complement system which occursthrough binding of an antibody to an antigen. These activities are knownto vary depending on a subclass of the antibody, which has been found tobe due to a structural difference in the constant region of antibodies(Charles A. Janeway et al., Immunology, 1997, Current BiologyLtd./Garland Publishing Inc.).

Anti-CD40 agonistic antibodies will be more preferable as therapeuticagent if they have not the activities of ADCC and/or CDC which mayinduce cell death of CD40-expressing cells, in terms of mechanism ofimmunoactive action. If CD40-expressing cells are injured by ADCC and/orCDC activities, immunosuppression may occur rather than desiredimmunoactivation, resulting in exacerbation of the disease. In addition,patients suffering from infectious diseases may have higher ADCC and/orCDC activities. Therefore, when such antibodies are applied toinfectious diseases, it is necessary to evaluate them for safety morecarefully, for example, using more active rabbit complements than thosepresent in healthy human serum or peripheral blood which could not beeffective to detect the above activities in this situation. Accordingly,mutants and recombinants were created which had no activity of ADCC orCDC and examined for their activity.

Since ADCC and/or CDC activities are known to vary depending on asubclass of the antibody of interest, conversion of the subclass mayreduce ADCC and/or CDC activities. For the human IgG subclasses, forexample, IgG4 is generally known to be a subclass with low activities ofboth ADCC and CDC, and it is reported that IgG2 is CDC active but poorlyactive in ADCC, while IgG1 is highly active in both ADCC and CDC(Charles A. Janeway et al., Immunology, 1997, Current BiologyLtd./Garland Publishing Inc.). Selection of a particular subclass bytaking advantage of the above characteristics may create a lesscytotoxic antibody from the original antibody. A combination of aspecific subclass of antibody with such a point mutation as describedbelow may create an antibody with a desired activity. Further, reductionin ADCC and/or CDC activities of an antibody is reported to be attainedby incorporation of a mutation into its constant region. For instance,L235, D265, D270, K322, P331, and P329 (each alphabetical letter denotesan amino acid by the single-letter notation, and each number denotes anEU index proposed by Kabat et al. (Kabat et. al., Sequences of proteinsof Immunological Interest, 1991 Fifth edition); such symbols will beused hereinafter) may play an important role in complement activation byhuman IgG, and substitution of one of those sites by another amino acidmay reduce the CDC activity. Esohe E. Idusogie et. al. J. Immunol. 2000,164:4178-4184, Yuanyuan Xu et. al. J. Biol. Chem. 1994, 269:3469-3474,Brekke, O. H. et. al. Eur. J. Immunol. 1994, 24:2542, Morgan, A., et.al., Immunology 1995, 86:319, Lund, J., et. al., J. Immunol., 1996,157:4963, Tao, M. H., et. al., J. Exp. Med. 1993, 178:661).Specifically, substitution of D270, K322, P329, or P331 by A may reducethe CDC activity. Substitution of P331 by S or G may also induce thesame thing.

It is believed that Glu233-Ser239, Gly316-Lys338, Lys274-Arg301,Tyr407-Arg416, Asn297, Glu318, Leu234-Ser239, Asp265-Glu269,Asn297-Thr299 and Ala327-Ile332 take a part in binding of IgG to FcR(Duncan, A. R., Woof, J. M., Partridge, L. J., Burton, D. R., andWinter, G. (1988) Nature 332, 563-564, Gessner, J. E., Heiken, H., Tamm,A., and Schmidt, R. E. (1998) Ann. Hematol. 76, 231-248, Gavin, A.,Hulett, M, and Hogarth, P. M. (1998) in The Immunoglobulin Receptors andTheir Physiological and Pathological Roles in Immunity (van de Winkel,J. G. J., and Hogarth, P. M., eds), pp. 11-35, Kluwer AcademicPublishers Group, Dordrecht, The Netherlands, Sautes, C. (1997) inCell-mediated Effects of Immunoglobulins (Fridman, W. H., and Sautes,C., eds), pp. 29-66, R. G. Landes Co., Austin, Tex., Da'ron, M. (1997)Annu. Rev. Immunol. 15, 203-234, Canfield, S. M., and Morrison, S. L.(1991) J. Exp. Med. 173, 1483-1491, Chappel, M. S., Isenman, D. E.,Everett, M., Xu, Y.-Y., Dorrington, K. J., and Klein, M. H. (1991) Proc.Natl. Acad. Sci. U.S.A. 88, 9036-9040, Woof, J. M., Partridge, L. J.,Jefferis, R., and Burton, D. R. (1986) Mol. Immunol. 23, 319-330, Wines,B. D., Powell, M. S., Parren, P. W. H. I., Barnes, N., and Hogarth, P.M. (2000) J. Immunol. 164, 5313-5318), and thus incorporation of amutation into one of these regions may reduce the ADCC activity.Specifically, substitution of L235 by E or G237 by A can reduce bindingof IgG to FcR.

The antibody according to the present invention has at least onemutation of amino acids to reduce the ADCC and/or CDC activities,preferably 1-20, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9,1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2 mutations.

The present invention has revealed that in some anti-CD40 antibodies,the hinge region of IgG2 is important in expression of their strongagonistic activities. Replacement of the variable region or the constantregion except the hinge region by a counterpart of any differentsubclass, or incorporation of a point mutation thereinto is expected tonot only modulate the ADCC and/or CDC activities, but increase theproductivity of the antibody, its stability during purification andstorage, and its blood kinetics.

To produce an antibody drug, the stability of the antibody duringpurification and storage is very important. Since the antibodies so fardeveloped belong mainly to the IgG1 subclass, conversion of the variableregion or the constant region except the hinge region to a sequencederived from the IgG1 subclass will be also effective to improve thephysical properties of the anti-CD40 agonistic antibodies describedabove.

The present invention provides mutants of agonistic anti-CD40 antibodiesand others as follows:

[1] A heavy chain of a monoclonal antibody having an agonistic activity,which binds to CD40, wherein the heavy chain comprises an upper hingeand a middle hinge derived from a human IgG2, and a constant region withat least one amino acid deleted or substituted, or with at least oneamino acid added thereto, said deletion, substitution or addition beingcapable of increasing or decreasing ADCC and/or CDC.[2] The heavy chain according to [1], wherein the constant region isderived from a human IgG.[3] The heavy chain according to [2], wherein the human IgG is a humanIgG1.[4] The heavy chain according to [2], wherein the human IgG is a humanIgG2.[5] The heavy chain according to [2], wherein the human IgG is a humanIgG3.[6] The heavy chain according to [2], wherein the human IgG is a humanIgG4.[7] The heavy chain according to any of [3] to [5], wherein saidsubstitution of amino acids in the constant region is substitution ofproline with serine at position 331 which is indicated by the EU indexas in Kabat et al.[8] A monoclonal antibody comprising the heavy chain according to any of[1] to [7].[9] The heavy chain according to any of [1] to [7], wherein the heavychain comprises a variable region from a heavy chain of a monoclonalantibody produced by the hybridoma KM341-1-19 (Accession No. FERMBP-7759).[10] A monoclonal antibody consisting of the heavy chain according to[9] and a light chain comprising a variable region from a light chain ofa monoclonal antibody produced by the hybridoma KM341-1-19 (AccessionNo. FERM BP-7759).[11] The heavy chain according to any of [1] to [7], wherein the heavychain comprises a variable region of the polypeptide represented by SEQID NO: 38.[12] A monoclonal antibody consisting of the heavy chain according to[11] and a light chain of a monoclonal antibody, wherein the light chaincomprises a variable region of the polypeptide represented by SEQ ID NO:40.[13] The heavy chain according to [1], wherein the heavy chain consistsof a remaining portion provided by removing the signal sequence from thepolypeptide represented by SEQ ID NO: 132.[14] A monoclonal antibody consisting of the heavy chain according to[13] and a light chain of a monoclonal antibody, wherein the light chainconsists of a remaining portion provided by removing the signal sequencefrom the polypeptide represented by SEQ ID NO: 134.[15] The heavy chain according to [1], wherein the heavy chain isproduced by a host comprising an expression vector having thepolynucleotide represented by SEQ ID NO: 131.[16] The monoclonal antibody according to [8], wherein the monoclonalantibody is produced by a host comprising an expression vector havingthe polynucleotide represented by SEQ ID NO: 131 and the polynucleotiderepresented by SEQ ID NO: 133.[17] The heavy chain according to any of [1] to [7], wherein the heavychain comprises a variable region from a heavy chain of a monoclonalantibody produced by the hybridoma 2105 (Accession No. FERM BP-8024).[18] A monoclonal antibody consisting of the heavy chain according to[17] and a light chain comprising a variable region from a light chainof a monoclonal antibody produced by the hybridoma 2105 (Accession No.FERM BP-8024).[19] The heavy chain according to any of [1] to [7], wherein the heavychain comprises a variable region of the polypeptide represented by SEQID NO: 42.[20] A monoclonal antibody consisting of the heavy chain according to[19] and a light chain of a monoclonal antibody, wherein the light chaincomprises a variable region of the polypeptide represented by SEQ ID NO:44.[21] The heavy chain according to [1], wherein the heavy chain consistsof a remaining portion provided by removing the signal sequence from thepolypeptide represented by SEQ ID NO: 136.[22] A monoclonal antibody consisting of the heavy chain according to[21] and a light chain of a monoclonal antibody, wherein the light chainconsists of a remaining portion provided by removing the signal sequencefrom the polypeptide represented by SEQ ID NO: 138.[23] The heavy chain according to [1], wherein the heavy chain isproduced by a host comprising an expression vector having thepolynucleotide represented by SEQ ID NO: 135.[24] The monoclonal antibody according to [8], wherein the monoclonalantibody is produced by a host comprising an expression vector havingthe polynucleotide represented by SEQ ID NO: 135 and the polynucleotiderepresented by SEQ ID NO: 137.[25] A polynucleotide represented by SEQ ID NO: 131.[26] A polynucleotide represented by SEQ ID NO: 133.[27] An expression vector having the polynucleotide according to [25].[28] An expression vector having the polynucleotide according to [26].[29] An expression vector having the polynucleotides according to [25]and [26].[30] A host comprising the expression vector according to [27].[31] A host comprising the expression vector according to [28].[32] A host comprising the expression vector according to [29].[33] A process of producing a heavy chain of a monoclonal antibody,comprising the steps of: culturing the host according to [30] in aculture medium; and obtaining a heavy chain of a monoclonal antibodyfrom the culture and/or the host.[34] A process of producing a monoclonal antibody, comprising the stepsof: culturing the host according to [32] in a culture medium; andobtaining a monoclonal antibody from the culture and/or the host.[35] A polynucleotide represented by SEQ ID NO: 135.[36] A polynucleotide represented by SEQ ID NO: 137.[37] An expression vector having the polynucleotide according to [35].[38] An expression vector having the polynucleotide according to [36].[39] An expression vector having the polynucleotides according to [35]and [36].[40] A host comprising the expression vector according to [37].[41] A host comprising the expression vector according to [38].[42] A host comprising the expression vector according to [39].[43] A process of producing a heavy chain of a monoclonal antibody,comprising the steps of: culturing the host according to [40] in aculture medium; and obtaining a heavy chain of a monoclonal antibodyfrom the culture and/or the host.[44] A process of producing a a monoclonal antibody, comprising thesteps of: culturing the host according to [42] in a culture medium; andobtaining a monoclonal antibody from the culture and/or the host.[45] A process of producing a heavy chain of a monoclonal antibodyhaving an agonistic activity capable of binding to CD40, comprising thestep of substituting the upper hinge and the middle hinge of anantibody, which is not either an upper hinge or a middle hinge derivedfrom a human IgG2, with an upper hinge and a middle hinge derived from ahuman IgG2, respectively.[46] A process of producing a heavy chain of a monoclonal antibodycomprising a variable region, and an upper hinge and a middle hingederived from a human IgG2, comprising the step of identifying apolypeptide forming the variable region, which is from a heavy chain ofa monoclonal antibody capable of binding to CD40.[47] A process of producing a monoclonal antibody having an agonisticactivity capable of binding to CD40, comprising the step of substitutingthe upper hinge and the middle hinge of an antibody, which is not eitheran upper hinge or a middle hinge derived from a human IgG2, with anupper hinge and a middle hinge derived from a human IgG2, respectively.[48] A process of producing a monoclonal antibody comprising a variableregion, and an upper hinge and a middle hinge derived from a human IgG2,comprising the step of identifying a polypeptide forming the variableregion, which is from a heavy chain of a monoclonal antibody capable ofbinding to CD40.[49] A pharmaceutical composition comprising the monoclonal antibodyaccording to [8], [10], [12], [14], [16], [18], [20], [22] or [24] as anactive ingredient.[50] The pharmaceutical composition according to [49] used forprevention or treatment of a malignant tumor, a pathogen or anautoimmune disease.[51] A method of prevention or treatment of a malignant tumor, apathogen or an autoimmune disease, comprising administration of thepharmaceutical composition according to [49] into a mammal.[52] Use of the monoclonal antibody according to [8], [10], [12], [14],[16], [18], [20], [22] or [24] for production of a pharmaceuticalcomposition used for prevention or treatment of a malignant tumor, apathogen or an autoimmune disease.[89] A polynucleotide provided by removing the portion encoding thesignal sequence from the polynucleotide represented by SEQ ID NO: 131.[90] A polynucleotide provided by removing the portion encoding thesignal sequence from the polynucleotide represented by SEQ ID NO: 133.[91] A polynucleotide provided by removing the portion encoding thesignal sequence from the polynucleotide represented by SEQ ID NO: 135.[92] A polynucleotide provided by removing the portion encoding thesignal sequence from the polynucleotide represented by SEQ ID NO: 137.

The present invention provides an antibody produced by modification ofan agonistic anti-CD40 antibody belonging to the human IgG2, wherein themodified antibody is a mutant having the constant region, exclusive ofthe upper and middle hinges, substituted with a sequence derived from adifferent subclass. The subclass is preferably IgG1. The presentinvention provides an antibody produced by modification of an agonisticanti-CD40 antibody belonging to the human IgG2, wherein the modifiedantibody is a mutant having the constant region, exclusive of the hingeregion, substituted with a sequence derived from a different subclass.The subclass is preferably IgG1.

Herein, reduction in ADCC and CDC activities means reduction in thoseactivities as compared with the corresponding activities of an anti-CD40monoclonal antibody other than the mutants described above, for example,as compared with the corresponding activities of a monoclonal antibodyproduced by the hybridoma KM341-1-19 (Accession No. FERM BP-7759) or2105 (Accession No. FERM BP-8024). The ADCC and CDC activities may beassayed by any known method, for example, the method described in theExamples herein. The sequences of variable regions in the heavy andlight chains of a monoclonal antibody will be presented below which isproduced by the hybridoma KM341-1-19 (Accession No. FERM BP-7759) or2105 (Accession No. FERM BP-8024).

DNA encoding variable regions in the heavy and light chains of theKM341-1-19 antibody and the amino acid sequences of the heavy and lightchains will be presented below.

In the heavy chain nucleotide sequence (SEQ ID NO: 37) of the KM341-1-19antibody, the signal sequence is initiated with adenine (A) at position50. The boundary between the signal sequence and the variable region islocated between “adenine” ([A]) at position 109 and cytosine (C) atposition 110, and the boundary between the variable region and theconstant region is located between adenine (A) at position 493 andguanine (G) at position 494 (the gene prediction software (Signal P ver.2) was used).

In the heavy chain amino acid sequence (SEQ ID NO: 38) of the KM341-1-19antibody, the boundary between the signal sequence and the variableregion is located between serine (S) at position 20 and glutamine (Q) atposition 21, and the boundary between the variable region and theconstant region is located between serine (S) at position 148 andalanine (A) at position 149.

Accordingly, the variable region in the heavy chain of the KM341-1-19antibody has a nucleotide sequence ranging from cytosine (C) at position110 to adenine (A) at position 493, as seen in SEQ ID NO: 37. Further,the variable region in the heavy chain of the KM341-1-19 antibody has anamino acid sequence ranging from glutamine (Q) at position 21 to serine(S) at position 148, as seen in SEQ ID NO: 38.

In the light chain nucleotide sequence (SEQ ID NO: 39) of the KM341-1-19antibody, the signal sequence is initiated with adenine (A) at position29. The boundary between the signal sequence and the variable region islocated between “adenine” ([A]) at position 88 and guanine (G) atposition 89, and the boundary between the variable region and theconstant region is located between adenine (A) at position 400 and“cytosine” ([C]) at position 401 (the gene prediction software (Signal Pver. 2) was used).

In the light chain amino acid sequence (SEQ ID NO: 40) of the KM341-1-19antibody, the boundary between the signal sequence and the variableregion is located between glycine (G) at position 20 and glutamic acid(E) at position 21, and the boundary between the variable region and theconstant region is located between lysine (K) at position 124 and“arginine” ([R]) at position 125.

Accordingly, the variable region in the light chain of the KM341-1-19antibody has a nucleotide sequence ranging from guanine (G) at position89 to adenine (A) at position 400, as seen in SEQ ID NO: 39. Further,the variable region in the light chain of the KM341-1-19 antibody has anamino acid sequence ranging from glutamic acid (E) at position 21 tolysine (K) at position 124, as seen in SEQ ID NO: 40.

The heavy chain nucleotide sequence (SEQ ID NO: 37) of the KM341-1-19antibody:

GTCGACGCTGAATTCTGGCTGACCAGGGCAGCCACCAGAGCTCCAGACAATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACAGGTCCAACTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTACTTGGAACTGGATCAGGCAGTCCCCATCGAGAGACCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATCGTGATTATGTAGGATCTGTGAAAAGTCGAATAATCATCAACCCAGACACATCCAACAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTATATATTACTGTACAAGAGCACAGTGGCTGGGAGGGGATTACCCCTACTACTACAGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCAGACCGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGATCCThe heavy chain amino acid sequence (SEQ ID NO: 38) of the KM341-1-19antibody:

MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRDLEWLGRTYYRSKWYRDYVGSVKSRIIINPDTSNNQFSLQLNSVTPEDTAIYYCTRAQWLGGDYPYYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKThe light chain nucleotide sequence (SEQ ID NO: 39) of the KM341-1-19antibody:

ACTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGThe light chain amino acid sequence (SEQ ID NO: 40) of the KM341-1-19antibody:

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNTFGPGTKVDIKRT

DNA encoding variable regions in the heavy and light chains of the 2105antibody and the amino acid sequences of the heavy and light chains willbe presented below.

In the heavy chain nucleotide sequence (SEQ ID NO: 41) of the 2105antibody, the signal sequence is initiated with adenine (A) at position70. The boundary between the signal sequence and the variable region islocated between “thymine” ([T]) at position 126 and guanine (G) atposition 127, and the boundary between the variable region and theconstant region is located between adenine (A) at position 495 andguanine (G) at position 496 (the gene prediction software (Signal P ver.2) was used).

In the heavy chain amino acid sequence (SEQ ID NO: 42) of the 2105antibody, the boundary between the signal sequence and the variableregion is located between cysteine (C) at position 19 and glutamic acid(E) at position 20, and the boundary between the variable region and theconstant region is located between serine (S) at position 142 andalanine (A) at position 143.

Accordingly, the variable region in the heavy chain of the 2105 antibodyhas a nucleotide sequence ranging from guanine (G) at position 127 toadenine (A) at position 495, as seen in SEQ ID NO: 41. Further, thevariable region in the heavy chain of the 2105 antibody has an aminoacid sequence ranging from glutamic acid (E) at position 20 to serine(S) at position 142, as seen in SEQ ID NO: 42.

In the light chain nucleotide sequence (SEQ ID NO: 43) of the 2105antibody, the signal sequence is initiated with adenine (A) at position28. The boundary between the signal sequence and the variable region islocated between “adenine” ([A]) at position 87 and guanine (G) atposition 88, and the boundary between the variable region and theconstant region is located between adenine (A) at position 405 and“cytosine” ([C]) at position 406 (the gene prediction software (Signal Pver. 2) was used).

In the light chain amino acid sequence (SEQ ID NO: 44) of the 2105antibody, the boundary between the signal sequence and the variableregion is located between glycine (G) at position 20 and glutamic acid(E) at position 21, and the boundary between the variable region and theconstant region is located between lysine (K) at position 126 and“arginine” ([R]) at position 127.

Accordingly, the variable region in the light chain of the 2105 antibodyhas a nucleotide sequence ranging from guanine (G) at position 88 toadenine (A) at position 405, as seen in SEQ ID NO: 43. Further, thevariable region in the light chain of the 2105 antibody has an aminoacid sequence ranging from glutamic acid (E) at position 21 to lysine(K) at position 126, as seen in SEQ ID NO: 44.

The heavy chain nucleotide sequence (SEQ ID NO: 41) of the 2105antibody:

CTGAACACAGACCCGTCGACTCCCAGGTGTTTCCATTCAGTGATCAGCACTGAACACAGAGGACTCACCATGGAGTTGGGACTGAGCTGGATTTTCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCTTGGTGCATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAGAGATAGGCTATTTCGGGGAGTTAGGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGThe heavy chain amino acid sequence (SEQ ID NO: 42) of the 2105antibody:

MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSLVHADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDRLFRGVRYYGMDVWGQGTTVTVSSASTKThe light chain nucleotide sequence (SEQ ID NO: 43) of the 2105antibody:

CTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCCACTGGCTCACTTTCGGCGGGGGGACCAAGGTGGAGATCAAACGTACGGTGThe light chain amino acid sequence (SEQ ID NO: 44) of the 2105antibody:

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATISCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSHWLTFGGGTKVEIKRTV

In the heavy chain nucleotide sequence (SEQ ID NO: 131) of the 341G2Ser,the boundary between the signal sequence and the variable region islocated between “adenine” ([A]) at position 60 and cytosine (C) atposition 61, and the boundary between the variable region and theconstant region is located between adenine (A) at position 444 andguanine (G) at position 445 (the gene prediction software (Signal P ver.2) was used).

In the heavy chain amino acid sequence (SEQ ID NO: 132) of the 341G2Ser,the boundary between the signal sequence and the variable region islocated between serine (S) at position 20 and glutamine (Q) at position21, and the boundary between the variable region and the constant regionis located between serine (S) at position 148 and alanine (A) atposition 149.

Accordingly, the variable region in the heavy chain of the 341G2Ser hasa nucleotide sequence ranging from cytosine (C) at position 61 toadenine (A) at position 444, as seen in SEQ ID NO: 131. Further, thevariable region in the heavy chain of the 341G2Ser has an amino acidsequence ranging from glutamine (Q) at position 21 to serine (S) atposition 148, as seen in SEQ ID NO: 132.

The entire heavy chain nucleotide sequence of the 341G2Ser (SEQ ID NO:131):

ATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACAGGTCCAACTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTACTTGGAACTGGATCAGGCAGTCCCCATCGAGAGACCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATCGTGATTATGTAGGATCTGTGAAAAGTCGAATAATCATCAACCCAGACACATCCAACAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGGCTATATATTACTGTACAAGAGCACAGTGGCTGGGAGGGGATTACCCCTACTACTACAGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGTGGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACCGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAThe entire heavy chain amino acid sequence of the 341G2Ser (SEQ ID NO:132):

MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRDLEWLGRTYYRSKWYRDYVGSVKSRIIINPDTSNNQFSLQLNSVTPEDTAIYYCTRAQWLGGDYPYYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPASIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In the light chain nucleotide sequence (SEQ ID NO: 133) of the 341G2Ser,the boundary between the signal sequence and the variable region islocated between “adenine” ([A]) at position 60 and guanine (G) atposition 61, and the boundary between the variable region and theconstant region is located between adenine (A) at position 372 and“cytosine” ([C]) at position 373 (the gene prediction software (Signal Pver. 2) was used).

In the light chain amino acid sequence (SEQ ID NO: 134) of the 341G2Ser,the boundary between the signal sequence and the variable region islocated between glycine (G) at position 20 and glutamic acid (E) atposition 21, and the boundary between the variable region and theconstant region is located between lysine (K) at position 124 and“arginine” ([R]) at position 125. Accordingly, the variable region inthe light chain of the 341G2Ser has a nucleotide sequence ranging fromguanine (G) at position 61 to adenine (A) at position 372, as seen inSEQ ID NO: 133. Further, the variable region in the light chain of the341G2Ser has an amino acid sequence ranging from glutamic acid (E) atposition 21 to lysine (K) at position 124, as seen in SEQ ID NO: 134.

The entire light chain nucleotide sequence of the 341G2Ser (SEQ ID NO:133):

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAThe entire light chain amino acid sequence of the 341G2Ser (SEQ ID NO:134):

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In the heavy chain nucleotide sequence (SEQ ID NO: 135) of the2105G2Ser, the boundary between the signal sequence and the variableregion is located between “thymine” ([T]) at position 57 and guanine (G)at position 58, and the boundary between the variable region and theconstant region is located between adenine (A) at position 426 andguanine (G) at position 427 (the gene prediction software (Signal P ver.2) was used).

In the heavy chain amino acid sequence (SEQ ID NO: 136) of the2105G2Ser, the boundary between the signal sequence and the variableregion is located between cysteine (C) at position 19 and glutamic acid(E) at position 20, and the boundary between the variable region and theconstant region is located between serine (S) at position 142 andalanine (A) at position 143.

Accordingly, the variable region in the heavy chain of the 2105G2Ser hasa nucleotide sequence ranging from guanine (G) at position 58 to adenine(A) at position 426, as seen in SEQ ID NO: 135. Further, the variableregion in the heavy chain of the 2105G2Ser has an amino acid sequenceranging from glutamic acid (E) at position 20 to serine (S) at position142, as seen in SEQ ID NO: 136.

The entire heavy chain nucleotide sequence of the 2105G2Ser (SEQ ID NO:135):

ATGGAGTTGGGACTGAGCTGGATTTTCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTIGGAATAGTGGTAGCTTGGTGCATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAGAGATAGGCTATTTCGGGGAGTTAGGTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAThe entire heavy chain amino acid sequence of the 2105G2Ser (SEQ ID NO:136):

MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSLVHADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDRLFRGVRYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPASIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In the light chain nucleotide sequence (SEQ ID NO: 137) of the2105G2Ser, the boundary between the signal sequence and the variableregion is located between “adenine” ([A]) at position 60 and guanine (G)at position 61, and the boundary between the variable region and theconstant region is located between adenine (A) at position 378 and“cytosine” ([C]) at position 379 (the gene prediction software (Signal Pver. 2) was used).

In the light chain amino acid sequence (SEQ ID NO: 138) of the2105G2Ser, the boundary between the signal sequence and the variableregion is located between glycine (G) at position 20 and glutamic acid(E) at position 21, and the boundary between the variable region and theconstant region is located between lysine (K) at position 126 and“arginine” ([R]) at position 127. Accordingly, the variable region inthe light chain of the 2105G2Ser has a nucleotide sequence ranging fromguanine (G) at position 61 to adenine (A) at position 378, as seen inSEQ ID NO: 137. Further, the variable region in the light chain of the2105G2Ser has an amino acid sequence ranging from glutamic acid (E) atposition 21 to lysine (K) at position 126, as seen in SEQ ID NO: 138.

The entire light chain nucleotide sequence of the 2105G2Ser (SEQ ID NO:137):

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGACCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCCACTGGCTCACTTTCGGCGGGGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAThe entire light chain amino acid sequence of the 2105G2Ser (SEQ ID NO:138):

MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSHWLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

2. Modification of Antagonistic Antibodies

Anti-CD40 antagonistic antibodies will be more preferable as therapeuticagent as well as the agonistic antibodies, if they have not theactivities of ADCC and/or CDC, in terms of mechanism of action.Furthermore, it is important that anti-CD40 antagonistic antibodies haveno activity to induce signals by their in vivo crosslinking via Fcreceptors, even if the ADCC activity cannot be detected. In other words,it is necessary to confirm that they are not activate the immunity, andsuch active antibodies may be desired as pharmaceutical agent. Anti-CD40antagonistic antibodies are promising as therapeutic agent for treatingautoimmune diseases or suppressing rejection in organ transplantation.If they induce an agonistic activity due to some effect after they areadministered to patients, however weak it may be, the symptoms mayworsen in contrast to the desired therapeutic effect. Thus, an antibodywithout any agonistic activity is more preferable as pharmaceuticalagent. In the present invention, incorporation of a point mutation L235E(means substitution of L at position 235 with E; similar symbols will beused hereinafter) into IgG4 has been demonstrated to be effective for invivo reduction in the agonistic activity, in the animal test usingmonkeys. Although IgG4 is a subclass with low activities of ADCC andCDC, it is reported that when it was attempted to express IgG4 asrecombinant protein in cells like CHO, its half-molecules were secreteddue to a poor S—S bonding between the heavy chains (Rob C. Aalberse etal., Immunology, 105, 9-19, 2002). To overcome this problem,incorporation of a mutation into the constant region of antibodies isreported to successfully promote the formation of the S—S bonding.Therefore, this type of mutation was also evaluated for its usefulness.Specifically, the mutation of substituting S at position 228 with P wasincorporated (S. Angal et al., Molecular Immunology, vol. 30, no. 1,105-108, 1993).

For antagonistic antibodies as well as agonistic antibodies, thestability of the antibody during purification and storage is veryimportant. There may be some methods to create such antibodies as arephysically better while keeping the antagonistic activity. The antibodypharmaceuticals so far offered commercially belong mostly to the IgG1subclass, and they are not reported to be problematic in pharmaceuticalformulation. Based on these facts, it may be advantageous from theviewpoint of physical properties to derive the constant region ofantibodies from IgG1. In case of anti-CD40 antibodies, however, they aredesirably lower in ADCC and CDC activities. As a consequence, antibodieshaving an IgG1-type constant region modified with some point mutationsmay be desired. The mutations described above are useful to create suchantibodies. The IgG1-type constant region may become lower in ADCC andCDC activities by incorporating point mutation P331G thereinto. It isalso observed that incorporation of point mutation L235E into IgG4eliminates a slight agonistic activity in vivo to make itpharmaceutically more active, but makes it physically less stable at alow pH. Thus, substitution of L235 with an amino acid other than E maymake it physically more functional. As to the 4D11 antibody, it is verysimilar to the 2B 11 antibody with respect to the structure of itsvariable region. The 2B11 antibody has a lower antagonistic activity,but has a higher stability at a low pH, compared with the 4D11 antibody.If some amino acids derived from the constant region of 2B11 areincorporated into the 4D11 antibody, based on the above properties, 4D11may become more stable. Specifically, point mutation L38V, P58R, G62W,I79M, K81Y, H87Y, S98A, K109R, V120M or T124A in the heavy chain, orN75S in the light chain, or a combination thereof may be effective forthat purpose. Specifically, a mutant created by substituting L atposition 38 in the variable region of the heavy chain of the 4D11antibody with V (abbreviated as L38V; similar symbols will be usedhereinafter), a P58R mutant, a G62W mutant, a I79M mutant, a K81Ymutant, a H87Y mutant, a S98A mutant, a K109R mutant, a V120M mutant ora T124A mutant, or a N75S mutant in the light chain, or a combinationthereof may be provided for that purpose.

The antibody according to the present invention has at least onemutation of amino acids to reduce the ADCC and/or CDC activities,preferably 1-15, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4,1-3, or 1 or 2 mutations.

The present invention provides mutants of antagonistic anti-CD40antibodies and others as follows:

[53] A heavy chain of a monoclonal antibody having an antagonisticactivity capable of binding to CD40, wherein the heavy chain comprises aconstant region with at least one amino acid deleted or substituted, orwith at least one amino acid added thereto, said deletion, substitutionor addition being capable of increasing or decreasing ADCC and/or CDC.[54] The heavy chain according to [53], wherein the constant region isderived from a human IgG.[55] The heavy chain according to [54], wherein the human IgG is a humanIgG1.[56] The heavy chain according to [54], wherein the human IgG is a humanIgG2.[57] The heavy chain according to [54], wherein the human IgG is a humanIgG3.[58] The heavy chain according to [54], wherein the human IgG is a humanIgG4.[59] The heavy chain according to any of [55], [57] or [58], whereinsaid substitution of amino acids in the constant region is substitutionof leucine with glutamic acid at position 235 which is indicated by theEU index as in Kabat et al.[60] A heavy chain according to any of [53] to [58], wherein the heavychain comprises a constant region with at least one amino acid deletedor substituted, or with at least one amino acid added thereto, saiddeletion, substitution or addition being capable of promoting theformation of the S—S bond between the heavy chains.[61] The antibody heavy chain according to [60], wherein saidsubstitution of amino acids in the constant region is substitution ofserine with proline at position 228 which is indicated by the EU indexas in Kabat et al.[62] A monoclonal antibody comprising the heavy chain according to anyof [53] to [61].[63] The heavy chain according to any of [53] to [61], wherein the heavychain comprises a variable region from a heavy chain of a monoclonalantibody produced by the hybridoma 4D11 (Accession No. FERM BP-7758).[64] A monoclonal antibody comprising the heavy chain according to [63]and a light chain comprising a variable region from a light chain of amonoclonal antibody produced by the hybridoma 4D11 (Accession No. FERMBP-7758).[65] The heavy chain according to any of [53] to [61], wherein the heavychain comprises a variable region of the polypeptide represented by SEQID NO: 46.[66] A monoclonal antibody consisting of the heavy chain according to[65] and a light chain of a monoclonal antibody, wherein the light chaincomprises a variable region of the polypeptide represented by SEQ ID NO:48.[67] The heavy chain according to [53], wherein the heavy chain consistsof a remaining portion provided by removing the signal sequence from thepolypeptide represented by SEQ ID NO: 140.[68] A monoclonal antibody consisting of the heavy chain according to[67] and a light chain of a monoclonal antibody, wherein the light chainconsists of a remaining portion provided by removing the signal sequencefrom the polypeptide represented by SEQ ID NO: 142.[69] The heavy chain according to [53], wherein the heavy chain isproduced by a host comprising an expression vector having thepolynucleotide represented by SEQ ID NO: 139.[70] The monoclonal antibody according to [62], wherein the monoclonalantibody is produced by a host comprising an expression vector havingthe polynucleotide represented by SEQ ID NO: 139 and the polynucleotiderepresented by SEQ ID NO: 141.[71] A polynucleotide represented by SEQ ID NO: 139.[72] A polynucleotide represented by SEQ ID NO: 141.[73] An expression vector having the polynucleotide according to [71].[74] An expression vector having the polynucleotide according to [72].[75] An expression vector having the polynucleotides according to [71]and [72].[76] A host comprising the expression vector according to [73].[77] A host comprising the expression vector according to [74].[78] A host comprising the expression vector according to [75].[79] A process of producing a heavy chain of a monoclonal antibody,comprising the steps of: culturing the host according to [76] in aculture medium; and obtaining a heavy chain of a monoclonal antibodyfrom the culture and/or the host.[80] A process of producing a monoclonal antibody, comprising the stepsof: culturing the host according to [78] in a culture medium; andobtaining a monoclonal antibody from the culture and/or the host.[81] A pharmaceutical composition comprising the monoclonal antibodyaccording to [62], [64], [66], [68] or [70] as an active ingredient.[82] The pharmaceutical composition according to [81] used forprevention or treatment of transplant rejection, an autoimmune disease,allergy or blood clotting factor VIII inhibition.[83] A method of prevention or treatment of transplant rejection, anautoimmune disease, allergy or blood clotting factor VIII inhibition,which comprises administering the pharmaceutical composition accordingto [81] into a mammal.[84] Use of the monoclonal antibody according to [62], [64], [66], [68]or [70] for production of a pharmaceutical composition used forprevention or treatment of transplant rejection, an autoimmune disease,allergy or blood clotting factor VIII inhibition.[85] A method of producing a heavy chain of a monoclonal antibody havingan antagonistic activity capable of binding to CD40, wherein theagonistic activity is lowered, comprising a step of making deletion orsubstitution of at least one amino acid, or addition of at least oneamino acid in a constant region of a heavy chain of a human antibody.[86] The method according to [85], wherein the constant region is from ahuman IgG.[87] The method according to [86], wherein the human IgG is a humanIgG4.[88] The method according to any of [85] to [87], wherein saidsubstitution of amino acids in the constant region is substitution ofleucine with glutamic acid at position 235 which is indicated by the EUindex as in Kabat et al.[93] A polynucleotide provided by removing the portion encoding thesignal sequence from the polynucleotide represented by SEQ ID NO: 139.[94] A polynucleotide provided by removing the portion encoding thesignal sequence from the polynucleotide represented by SEQ ID NO: 141.

Additionally, the present invention provides the materials below.

A mutant of an antagonistic anti-CD40 antibody, comprising at least onesubstitution selected from the group consisting of substitution of Lwith V at position 38, substitution of P with R at position 58,substitution of G with W at position 62, substitution of I with M atposition 79, substitution of K with Y at position 81, substitution of Hwith Y at position 87, substitution of S with A at position 98,substitution of K with Rat position 109, substitution of V with M atposition 120 and substitution of T with A at position 124, whichsubstitutions are all carried out in a variable region of a heavy chainof a monoclonal antibody produced by the hybridoma 4D11 (Accession No.FERM BP-7758), and a mutant of an antagonistic anti-CD40 antibodycomprising substitution of N with S at position 75 in a variable regionof a light chain of the 4D11 antibody.

Herein, reduction in ADCC and CDC activities means reduction in thoseactivities as compared with the corresponding activities of an anti-CD40monoclonal antibody other than the mutants described above, for example,as compared with the corresponding activities of a monoclonal antibodyproduced by the hybridoma 4D11 (Accession No. FERM BP-7758). The ADCCand CDC activities may be assayed by any known method, for example, themethod described in the Examples herein. The sequences of variableregions in the heavy and light chains of a monoclonal antibody will bepresented below which is produced by the hybridoma 4D11 (Accession No.FERM BP-7758).

DNA encoding variable regions in the heavy and light chains of the 4D11antibody and the amino acid sequences of the heavy and light chains willbe presented below, respectively.

In the heavy chain nucleotide sequence (SEQ ID NO: 45) of the 4D11antibody, the boundary between the signal sequence and the variableregion is located between “cytosine” ([C]) at position 93 and cytosine(C) at position 94, and the boundary between the variable region and theconstant region is located between adenine (A) at position 456 andguanine (G) at position 457 (the gene prediction software (Signal P ver.2) was used).

In the heavy chain amino acid sequence (SEQ ID NO: 46) of the 4D11antibody, the boundary between the signal sequence and the variableregion is located between serine (S) at position 26 and glutamine (Q) atposition 27, and the boundary between the variable region and theconstant region is located between serine (S) at position 147 andalanine (A) at position 148.

Accordingly, the variable region in the heavy chain of the 4D11 antibodyhas a nucleotide sequence ranging from cytosine (C) at position 94 toadenine (A) at position 456, as seen in SEQ ID NO: 45. Further, thevariable region in the heavy chain of the 4D11 antibody has an aminoacid sequence ranging from glutamine (Q) at position 27 to serine (S) atposition 147, as seen in SEQ ID NO: 46.

In the light chain nucleotide sequence (SEQ ID NO: 47) of the 4D11antibody, the boundary between the signal sequence and the variableregion is located between “thymine” ([T]) at position 124 and guanine(G) at position 125, and the boundary between the variable region andthe constant region is located between adenine (A) at position 442 and“cytosine” ([C]) at position 443 (the gene prediction software (Signal Pver. 2) was used).

In the light chain amino acid sequence (SEQ ID NO: 48) of the 4D11antibody, the boundary between the signal sequence and the variableregion is located between cytosine (C) at position 22 and alanine (A) atposition 23, and the boundary between the variable region and theconstant region is located between lysine (K) at position 128 and“arginine” ([R]) at position 129.

Accordingly, the variable region in the light chain of the 4D11 antibodyhas a nucleotide sequence ranging from guanine (G) at position 125 toadenine (A) at position 442, as seen in SEQ ID NO: 47. Further, thevariable region in the light chain of the 4D11 antibody has an aminoacid sequence ranging from alanine (A) at position 23 to lysine (K) atposition 128, as seen in SEQ ID NO: 48.

The heavy chain nucleotide sequence (SEQ ID NO: 45) of the 4D11antibody:

ATATGTCGACGAGTCATGGATCTCATGTGCAAGAAAATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTACTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGCGGCTCCATCAGCAGTCCTGGTTACTACGGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATAAAAGTGGGAGCACCTACCACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTACGAGACCTGTAGTACGATATTTTGGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCThe heavy chain amino acid sequence (SEQ ID NO: 46) of the 4D11antibody:

MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLLKPSETLSLTCTVSGGSISSPGYYGGWIRQPPGKGLEWIGSIYKSGSTYHNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTRPVVRYFGWFDPWGQGTLVTVSSASThe light chain nucleotide sequence (SEQ ID NO: 47) of the 4D11antibody:

AGATCTTAAGCAAGTGTAACAACTCAGAGTACGCGGGGAGACCCACTCAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAATTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGThe light chain amino acid sequence (SEQ ID NO: 48) of the 4D11antibody:

MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPTFGQGTKVEIKRT

In the heavy chain nucleotide sequence (SEQ ID NO: 139) of the 4D11antibody G4PE, the boundary between the signal sequence and the variableregion is located between “cytosine” ([C]) at position 78 and cytosine(C) at position 79, and the boundary between the variable region and theconstant region is located between adenine (A) at position 441 andguanine (G) at position 442 (the gene prediction software (Signal P ver.2) was used).

In the heavy chain amino acid sequence (SEQ ID NO: 140) of the 4D11antibody, the boundary between the signal sequence and the variableregion is located between serine (S) at position 26 and glutamine (Q) atposition 27, and the boundary between the variable region and theconstant region is located between serine (S) at position 147 andalanine (A) at position 148.

Accordingly, the variable region in the heavy chain of the 4D11 antibodyhas a nucleotide sequence ranging from cytosine (C) at position 79 toadenine (A) at position 441, as seen in SEQ ID NO: 139. Further, thevariable region in the heavy chain of the 4D11 antibody has an aminoacid sequence ranging from glutamine (Q) at position 27 to serine (S) atposition 147, as seen in SEQ ID NO: 140.

The entire heavy chain nucleotide sequence (SEQ ID NO: 139) of the4D11G4PE:

ATGGATCTCATGTGCAAGAAAATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTACTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGCGGCTCCATCAGCAGTCCTGGTTACTACGGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATAAAAGTGGGAGCACCTACCACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTACGAGACCTGTAGTACGATATTTTGGGTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGGCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCGAGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGAThe entire heavy chain amino acid sequence (SEQ ID NO: 140) of the4D11G4PE:

MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLLKPSETLSLTCTVSGGSISSPGYYGGWIRQPPGKGLEWIGSIYKSGSTYHNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTRPVVRYFGWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

In the light chain nucleotide sequence (SEQ ID NO: 141) of the 4D11G4PE,the boundary between the signal sequence and the variable region islocated between “thymine” ([T]) at position 66 and guanine (G) atposition 67, and the boundary between the variable region and theconstant region is located between adenine (A) at position 384 and“cytosine” ([C]) at position 385 (the gene prediction software (Signal Pver. 2) was used).

In the light chain amino acid sequence (SEQ ID NO: 142) of the 4D11G4PE,the boundary between the signal sequence and the variable region islocated between cytosine (C) at position 22 and alanine (A) at position23, and the boundary between the variable region and the constant regionis located between lysine (K) at position 128 and “arginine” ([R]) atposition 129.

Accordingly, the variable region in the light chain of the 4D11G4PE hasa nucleotide sequence ranging from guanine (G) at position 67 to adenine(A) at position 384, as seen in SEQ ID NO: 141. Further, the variableregion in the light chain of the 4D11 antibody has an amino acidsequence ranging from alanine (A) at position 23 to lysine (K) atposition 128, as seen in SEQ ID NO: 142.

The entire light chain nucleotide sequence (SEQ ID NO: 141) of the4D11G4PE:

ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAATTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGGTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGAThe entire light chain amino acid sequence (SEQ ID NO: 142) of the4D11G4PE:

MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

3. Definition

The terms used herein will be defined below.

“CD40” described in the present invention refers to a polypeptide havingthe amino acid sequence described in E. A. Clark et al., Proc. Natl.Acad. Sci. USA 83: 4494, 1986, or I. Stamenkovic et al., EMBO J. 8:1403, 1989, and particularly, an antigenic polypeptide expressed on thesurface of B cells, DC, macrophages, endothelial cells, epithelial cellsor tumor cells derived from them.

“An anti-CD40 antibody” refers to any monoclonal antibody to acell-expressed CD40, a full-length CD40 or a partial-length CD40.

In addition, “an antibody” of the invention is derived from genes(collectively called antibody genes) encoding a heavy chain variableregion and a heavy chain constant region, as well as a light chainvariable region and a light chain constant region which togetherconstitute an immunoglobulin. Human immunoglobulins are grouped into 5different classes consisting of IgG, IgA, IgM, IgD, and IgE. Further,IgG is composed of 4 different subclasses, IgG1, IgG2, IgG3 and IgG4,while IgA is composed of 2 different subclasses, IgA1 and IgA2. IgG1,IgG2, IgG3 and IgG4 are located in 14q32, 33 of the human chromosomes.The fundamental structure of immunoglobulin is composed of twohomologous L chains (light chains) and two homologous H chains (heavychains). The class and subclass of an immunoglobulin is determined byits H chains. The antibody according to the present invention maycomprise any class, any subclass or any isotype of immunoglobulin. “Afunctional fragment” of the inventive antibody refers to a portion(partial fragment) of the antibody defined above that is active singlyor multiply on an antigen to the antibody, including, for example,F(ab′)₂, Fab′, Fab, Fv, disulfide-stabilized FV, single-chain FV (scFV)and a multimer thereof (D. J. King, Applications and Engineering ofMonoclonal Antibodies, 1998, T. J. International Ltd.).

Up to now, it has been known that IgG1 includes J00228, Z17370 andY14737, IgG2 includes J00230, AJ250170, AF449616, AF449617, AF449618,Z49802 and Z49801, IgG3 includes M12958, K01313, X16110, X99549,AJ390236, AJ390237, AJ390238, AJ390241, AJ390242, AJ390246, AJ390247,AJ390252, AJ390244, AJ390254, AJ390260, AJ390262, AJ390272, AJ390276 andAJ390279, and IgG4 includes K01316, AJ001563 and AJ001564 (the symbolslisted above indicates accession numbers of the genes).

In the present invention, CH1, hinge, CH2 and CH3 each denote a portionof the heavy-chain constant region of any antibody, and are based on theEU index as in Kabat et al. (Kabat et al., Sequences of proteins ofimmunological interest, 1991 Fifth edition). By definition, CH1 rangesfrom 118 to 215 by the EU index, hinge ranges from 216 to 230 by the EUindex, CH2 ranges from 231 to 340 by the EU index, and CH3 ranges from341 to 446 by the EU index.

The “human antibody” of the present invention means an antibody which isan expression product of a human-derived antibody gene.

“Agonistic” refers to an action of enhancing binding of a ligand to CD40expressed on the surface of such cells as B cells, tumor cells ordendritic cells, or an action of providing the CD40-expressing cellswith at least one effect which the CD40 ligand makes on theCD40-expressing cells. “An agonistic antibody” refers to an antibodyhaving such an agonistic action. An example of the effects provided forthe CD40-expressing cells is to promote the expression of CD95.

“Antagonistic” refers to an action of inhibiting binding of the ligandto CD40 expressed on the surface of such cells as B cells, tumor cellsor dendritic cells, or an action of neutralizing at least one effectwhich the CD40 ligand makes on the CD40-expressing cells. “Anantagonistic antibody” refers to an antibody having such an antagonisticaction. An example of the effects provided for the CD40-expressing cellsis to suppress the proliferation of B cells or the production ofantibodies.

The present application clearly presents antibodies, or heavy chain orlight chain variable regions thereof by amino acid sequences. Thepresent invention also encompasses the amino acid sequences with atleast one amino acid deleted or substituted, or added thereto,preferably 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2 aminoacids.

The present application clearly presents genes which encode antibodies,or heavy chain or light chain variable regions thereof by nucleotidesequences. The present invention also encompasses the nucleotidesequences with at least one nucleotide deleted or substituted, or addedthereto, preferably 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2amino acids.

The anti-CD40 antibody according to the present invention can beprovided by incorporating an antibody gene into an expression vector,transfecting the vector into a suitable host cell, harvesting theantibody from the cultured cells or the supernatant, and purifying it.

The vector may be a phage or a plasmid which can replicate in the hostcell by itself or can be integrated into the chromosome of the hostcell. The plasmid DNA may be derived from Escherichia coli, Bacillussubtilis or a yeast, while the phage DNA may be from λ phage.

The host cell for transformation is not particularly limited if it canexpress the target gene. Examples of the host cell may include bacteria(Escherichia coli, Bacillus subtilis, etc.), yeasts, animal cells (COScell, CHO cell, etc.) and insect cells.

There are known modes of transferring the gene into the host cells,including any mode, such as mediation by calcium ion, electroporation,spheroplast fusion, mediation by lithium acetate, calcium phosphatetransfection or lipofection. In order to transfer the gene into ananimal, as described later, the modes include microinjection;electroporation or lipofection for ES cells; and nucleartransplantation.

In the present invention, “culture” refers to (a) culture supernatant,(b) cultured cells, cultured biomass or disrupted matter thereof, or (c)secretion of transformant. To culture the transformant, a mediumsuitable for the host is used and static culture, roller bottle cultureor something else may be employed.

After the culture, if the desired antibody protein is produced withinthe biomass or cells, the antibody is harvested by disrupting thebiomass or cells. If the desired antibody is produced out of the biomassor cells, the culture solution is used as it is or after it is separatedfrom the biomass or cells by centrifugation or other means. Thereafter,a biochemical process utilizing any chromatography, which is appropriatefor separation/purification of proteins, is employed alone or optionallyin combination with another to separate/isolate the desired antibodyfrom the culture.

Furthermore, the technology of creating a transgenic animal may be usedto produce a transgenic animal that is a host animal having the geneintegrated into an endogenous gene, such as a transgenic bovine, atransgenic goat, a transgenic sheep or a transgenic pig (Wright, G., etal., (1991) Bio/Technology 9, 830-834) and a large amount of amonoclonal antibody derived from the antibody gene can be obtained fromthe milk secreted from the transgenic animal. The culture of a hybridomain vitro can be done by using a known nutrient medium or any nutrientmedium derivatively prepared from known basic media as used to grow,maintain and store the hybridoma and to produce a monoclonal antibody inthe supernatant, depending on the properties of the cultured hybridoma,the purpose of the study and the culture method.

4. Antibody Properties (1) Agonistic Antibodies

The mutant of the agonistic antibody according to the present inventionmay activate the immune system without injuring immunocompetent cells,since it has an ADCC and/or CDC activity equal to or lower than theoriginal antibody, while keeping an agonistic activity. It is thusexpected that the mutant exhibits the immunoactivating action which isequal to or higher than the original antibody and the cytotoxicity toCD40-expressing cells which is equal to or lower than the originalantibody.

(2) Antagonistic Antibodies

The mutant of the antagonistic anti-CD40 antibody according to thepresent invention has the reduced ADCC and/or CDC activity compared tothe unmodified antibody, while keeping a suppressive activity againstimmunoactivating signals induced by CD40L. It is also expected todecrease the activity of signal induction in vivo which is considered tooccur via Fc receptors.

5. Pharmaceutical Compositions

A pharmaceutical composition containing a formulation of the purifiedantibody according to the present invention is also within the scope ofthe present invention. The pharmaceutical composition may preferablycontain a physiologically acceptable diluent or carrier in addition tothe antibody, and may be a mixture thereof with a different antibody ora different drug such as an antibiotic agent. The suitable carrier mayinclude, but not limited to, physiological saline, phosphate bufferedphysiological saline, phosphate buffered physiological saline glucosesolution and buffered physiological saline. Alternatively, the antibodymay be freeze-dried for storage and when it is used, reconstituted in anaqueous buffer as described above. The pharmaceutical composition may beadministered via the oral route, or the parenteral route, such asintravenous, intramuscular, subcutaneous or intraperitoneal injection ordosing.

A single effective dose, which is a combination of the antibody of thepresent antibody with a suitable diluent and a physiologicallyacceptable carrier, is from 0.0001 mg to 100 mg per kg of body weight,and it may be taken at a time interval of from 2 days to 8 weeks.

When the pharmaceutical composition of the present antibody is anagonistic antibody, it is used as: immunostimulant (antiviral oranti-infective agent) for pathogens including, for example, hepatitis A,B, C, D or E virus, HIV, influenza virus, simple herpes virus,cytomegalovirus, EB virus, papiloma virus, chlamydia, mycoplasma,toxoplasma, malaria, trypanosome and tubercle bacillus; antitumor agentfor malignant tumors having cancer cells with CD40 expressed, including,for example, pancreatic cancer, bladder cancer, lymphoma (e.g.,Hodgkin's lymphoma), leukemia, malignant melanoma, pancreatic cancer,lung cancer, ovarian cancer, bladder cancer, breast cancer, coloncancer, prostatic cancer, and head and neck cancer; and therapeuticagent for autoimmune diseases such as rheumatism. The pharmaceuticalcomposition may be used for a combination of the above diseases. It maybe also used in combination as adjuvant for a cancer-specific peptide.When the pharmaceutical composition is an antagonistic antibody, on theother hand, it is useful as: immunosuppressant in organ transplantation(preventive or therapeutic agent for rejection in transplantation ofpancreatic islets, kidney or something else, or GVHD), therapeutic agentfor autoimmune diseases (e.g., rheumatism, psoriasis, chronic ulcerativecolitis, Crohn's disease, systemic lupus erythematosus, multiplesclerosis, myasthenia, scleroderma, antiphospholipid antibodiessyndrome, autoimmune hepatitis, idiopathic thrombocytopenic purpura,Behcet's syndrome, arteriosclerosis, nephritis and respiratory distresssyndrome), therapeutic agent for allergy (e.g., asthma), and therapeuticagent for blood clotting factor VIII inhibition. The pharmaceuticalcomposition may be used for a combination of the above diseases.

6. Epitopes

The binding epitopes of CD40 were determined for the KM341-1-19 and 2105antibodies having a superior agonistic activity, and for the 4D11antibody having a superior antagonistic activity, respectively (Example2). The present invention provides antibodies having an agonistic orantagonistic activity which have a different variable region sequencefrom those of the above antibodies but recognize the same epitope as oneof the above antibodies. These antibodies can be obtained in such aprocedure as described below.

When it is intended to acquire an agonistic anti-CD40 antibodyrecognizing the same epitope as the KM341-1-19 antibody, for example,mice or the likes are immunized with CD40 to provide monoclonalantibodies, from which some monoclonal antibodies competing with theKM341-1-19 antibody to bind to CD40 are screened according to thestandard procedure. From the screened antibodies, an antibody having thesame pattern of binding to the peptide as the KM341-1-19 antibody isselected according to the method described in Example 2.

The present invention will be described in more detail below withreference to examples. However, the present invention is not limited toembodiments described in the examples.

Example 1 Expression and Purification of Antibody and Antigen Proteins

A vector plasmid containing a variable region of an antibody wastransfected into CHO cells (ATCC), and antibody-expressing cells wereselected by G418 to prepare a stable expression cell line.

A mutant antigen was expressed by transiently introducing a vector intoHEK cells (ATCC).

An anti-CD40 antibody was purified from the above culture supernatant bythe following method. The culture supernatant containing an anti-CD40antibody was affinity purified in a Hyper D® Protein A column(manufactured by NGK Insulators, Ltd.) or in case of mouse IgG1purification, a Protein G column (Amersham Pharmacia Biotech) accordingto the attached instruction using PBS(−) as an adsorption buffer and a0.1 M sodium citrate buffer (pH 3) as an elution buffer. The elutedfraction was adjusted to about pH 7.2 by addition of a 1 M Tris-HCl (pH8.0) or Na2HPO4 solution. The prepared antibody solution was substitutedwith PBS(−) using a dialysis membrane (10,000 cuts, manufactured bySpectrum Laboratories, Inc.) or an SP column (Amersham PharmaciaBiotech), and filtered and sterilized using a membrane filter MILLEX®-GVwith a pore diameter of 0.22 μm (manufactured by Millipore Corp.). Theconcentration of the purified antibody was calculated by measurement ofthe absorbance at 280 nm, taking 1 mg/ml as 1.450D.

Example 2 Determination of Epitopes

A 13-mer peptide covering amino acid 175 (SEQ ID NO: 1) in anextracellular region of CD40 was shifted by two amino acids each tosynthesize 82 peptides in total (SEQ ID NOS: 49 to 130) as spots fromthe C-terminal on a cellulose membrane and acetylate the N-terminalthereof (Jerini AG, Germany). The reaction thereafter was carried outbased on a conventional Western analysis (see Reineke, U. et al. (2001),“Epitope mapping with synthetic peptides prepared by SPOT synthesis.”Antibody Engineering (Springer Lab Manual) Eds.: Kontermann/Dubel,433-459, for example). In the analysis, coloring intensity of each spotwas quantified using LumiImager™ (Boehringer-Mannheim Corp.) (FIGS.1A-1, A-2, B-1 and B-2).

The results confirmed that an 4D11 antibody strongly recognizes the 20thto 24th and 41st peptides, a 2105 antibody strongly recognizes the 12thto 23rd and 64th peptides, a KM341-1-19 antibody strongly recognizes the41st and 42nd peptides, KM643-4-11 strongly recognizes the 43rd peptide,F72 strongly recognizes the 75th peptide, 110 strongly recognizes the64th peptide, F4-465 strongly recognizes the 34th, 35th, 54th, 55th,65th, 66th and 75 peptides, KM281-1-10 strongly recognizes the 21st,24th, 64th and 75th peptides, 2B11 (novel antibody) strongly recognizesthe 21st, 24th and 64th peptides, and F76 (novel antibody) stronglyrecognizes the 21st, 35th, 51st and 52nd peptides.

In order to confirm the binding site of the anti-CD40 antibody, aCD40-FC fusion protein having a mutation introduced thereinto wasprepared, and the binding ability thereto was examined by ELISA. Sincethe anti-CD40 antibody does not cross-react with mouse B cells, fiveCD40Fc fusion proteins were prepared by partially converting the aminoacid sequence into that of mouse CD40. Binding of the antibody to theantigens was examined. The method for preparing the mutant CD40-FCfusion proteins is shown below. The mutation site was prepared byintroducing a mouse CD40 sequence into a part to which the antibodystrongly binds of the peptide sequence. CD40mut1 converted EFTE at asite corresponding to the 15th peptide into ALEK, CD40mut2 converted LDTat a site corresponding to the 21st peptide into SAQ, CD40mut3 convertedTH at a site corresponding to the 24th peptide into IR, CD40mut4converted EEGW at a site corresponding to the 42nd peptide into KEGQ,and CD40mut5 converted VSSA at a site corresponding to the 64th peptideinto QSSL. The mutants were prepared according to a gene engineeringtechnique (FIGS. 2A, B and C). The analysis results confirmed that the2105 antibody has extremely reduced binding ability to CD40mut1. Theresults also confirmed that the 4D11 antibody and 2B11 have reducedbinding ability to CD40mut2.

Example 3 Binding Activity of Anti-CD40 Agonistic Antibody to RamosCells

A Ramos cell line was suspended in a PBS staining buffer (SB) containing0.1% NaN₃ and 2% FCS at a concentration of 2×10⁶/ml. The cell suspension(100 μl/well) was dispensed to a 96-well round-bottom plate(manufactured by Becton, Dickinson and Company). Each hybridoma culturesupernatant (50 μl) was added, and incubated at an ice temperature for30 minutes. A human IgG1 antibody to human serum albumin as a negativecontrol was adjusted to a concentration of 2 μg/ml in a hybridomaculture medium, added in an amount of 50 and then incubated at an icetemperature for 15 minutes. After washing the plate with SB, 50 μl of a250-fold diluted R-PE fluorescently labeled anti-human antibody(manufactured by Southern Biotechnology Associates, Inc.) was added, andincubated at an ice temperature for 15 minutes. After washing the platewith SB twice, it was suspended in 300 to 500 μl of a FACS buffer, andfluorescence intensity of each cell was measured using FACS (FACSort,FACScan, manufactured by Becton, Dickinson and Company).

Example 4 Evaluation of Agonistic Activity of Anti-CD40 AgonisticAntibody to Ramos Cells

5.0×10⁵ cells/ml of a Ramos cell suspension was seeded on a 96-wellplate at 100 μl/well. A hybridoma culture supernatant or purifiedantibody was diluted to 20 μg/ml in a medium, and the dilution was addedto the 96-well plate at a concentration of 100 μl/well. After culturingovernight, cells were harvested, and R-PE labeled anti-CD95 antibody(Pharmingen NJ) was used for the cells. Analysis was carried out usingFACScan or FACSsort (Becton, Dickinson and Company).

Example 5 Inhibition of CD95 Expression by Anti-CD40 AntagonisticAntibody in Ramos Cells

1.0×10⁶ cells/ml of a Ramos cell suspension was seeded on a 96-wellplate at 50 μl/well. A hybridoma culture supernatant or purifiedantibody was adjusted to 2 μg/ml in a medium, and the medium was addedto the 96-well plate at 100 μl/well. 4 μg/ml of a soluble CD40 ligand(Alexis Corporation) and 4 μg/ml of an anti-FLAG antibody (M2, Sigma)were added to a medium, and the medium was added to a 96-well plate at50 μl/well. After culturing overnight, cells were harvested, and an R-PElabeled anti-CD95 antibody (Pharmingen NJ) was used for the cells.Analysis was carried out using FACS.

Example 6 Measurement of CDC Activity in Anti-CD40 Antibody

In the CDC assay, 2,000 Cr⁵¹-labeled target cells and a humanserum-derived complement (manufactured by Sigma Co.) or rabbitserum-derived complement (Cedarlane Laboratories Limited, Ontario,Canada) at a final concentration of 5% were cultured in a round-bottom96-well plate in a total volume of 200 μL together with the antibody atvarious concentrations at 37° C. in the presence of 5% CO₂ for twohours.

After culturing, the plate was centrifuged to cause the cells toprecipitate, and then 50 μL of the supernatant was transferred to a96-well plate including a powder scintillator (Lumaplate™-96:manufactured by Packard Instrument Co., Inc.) and dried at 55° C. for1.5 hours. After confirming that the plate was dried, it was coveredwith a special cover (TopSeal™-A: 96-well microplates: manufactured byPackard Instrument Co., Inc.), and the γ-ray dose was measured with ascintillation counter (TopCount: manufactured by Packard Instrument Co.,Inc.).

Example 7 Measurement of ADCC Activity of Anti-CD40 Antibody

As antibody-mediated cytotoxicity, cytotoxicity to target cells in thepresence of cells having killer activity such as NK cells or neutrophilsand an antibody (Antibody-Dependent Cellular Cytotoxicity, hereinafterADCC), and cytotoxicity to target cells in the presence of a complementand an antibody (Complement-Dependent Cytotoxicity, hereinafter CDC)were measured. hIgG was used as a control.

The measurement method is simply described as follows. Radioactivechromium (Cr⁵¹) was incorporated into the cytoplasm of the target cells,and the amount of Cr⁵¹ released in the culture solution by cell deathwas measured as a γ-ray dose.

Specifically, 10⁵ target cells of a Burkitt's lymphoma cell line Raji(ATCC CCL-86) were suspended in 15 μL of fetal calf serum (FCS). 50 μL(37 MBq/mL) of Cr⁵¹-labeled sodium chromate (manufactured byPerkinElmer, Inc.: hereinafter referred to as Cr⁵¹) was added to thesuspension, and the cells were cultured at 37° C. for one hour. Next, 10mL of a medium was added, and the medium was discarded bycentrifugation. This operation was repeated three times to remove Cr⁵¹not incorporated in the cells.

In the ADCC assay, 2,000 Cr⁵¹-labeled target cells and 200,000 healthyhuman peripheral blood mononuclear leukocytes obtained by the methoddescribed in Example 6 were cultured in a round-bottom 96-well plate(manufactured by Falcon) in a total volume of 200 μL together with theantibody at various concentrations at 37° C. in the presence of 5% CO₂for four hours.

After culturing, the plate was centrifuged to cause the cells toprecipitate, and then 50 μL of the supernatant was transferred to a96-well plate including a powder scintillator (Lumaplate™-96:manufactured by Packard Instrument Co., Inc.) and dried at 55° C. for1.5 hours. After confirming that the plate was dried, the plate wascovered with a special cover (TopSeal™-A: 96-well microplates:manufactured by Packard Instrument Co., Inc.), and the γ-ray dose wasmeasured with a scintillation counter (TopCount: manufactured by PackardInstrument Co., Inc.).

Example 8 Preparation and Activity Evaluation of Anti-CD40 AgonisticAntibody P331S Mutant

Gene cloning of anti-CD40 agonistic antibodies KM341-1-19 and 2105 isdescribed in WO 02/099186. It is reported that CDC activity is reducedby converting Pro at position 331 in the IgG2 constant region into Ser.To reduce CDC activity of the KM341-1-19 antibody and the 2105 antibody,a P331S mutation was introduced into the IgG2 constant region thereof.

The human IgG1 constant region of an antibody-expressing vectorN5KG1-Val Lark (IDEC Pharmaceuticals: hereinafter abbreviated to N5KG1)was substituted with human IgG2 to prepare N5KG2, and the Pro atposition 331 of IgG2 was converted into Ser to prepare a mutation. cDNAcloning of the IgG2 constant region was carried out by harvestingKM341-1-19 hybridoma by centrifugation, adding TRIZOL (Gibco BRL), andextracting total RNA according to the instruction. The antibody cDNAvariable region was cloned using a SMART RACE cDNA amplification kit ofClontech Laboratories, Inc. according to the attached instruction. 1ststrand cDNA was prepared using 5 μg of total RNA as a template. PCR wascarried out with tnIgG3Nhe: atatGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGC (SEQID NO: 2)G and tnIgG2Bam: atatggatccTCATTTACCCGGAGACAGGGAGAGGCTC (SEQID: 3) as primer sequences using a ZtaqPCR kit (Takara) in 30 cycleseach consisting of reaction at 98° C. for 1 second, at 55° C. for 30seconds and at 72° C. for 1 minute to amplify the gene. After thereaction, the amplified product was purified by a QIAGEN PCRpurification kit, digested with NheI and BamHI, and incorporated intoN5KG1 to confirm the sequence. This vector was defined as N5KG2.

N5KG2Ser (with Pro at position 331 converted into Ser) was prepared asfollows. Reaction at 98° C. for 1 second, at 60° C. for 30 seconds andat 72° C. for 30 seconds was carried out 15 times using N5KG2 as atemplate and primers IgG3Nhe: atatGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCG(SEQ ID NO: 4) and G2Ser2: GTTTTCTCGATGGAGGCTGGGAGGCC (SEQ ID NO: 5). Atthe same time, reaction at 98° C. for 1 second, at 60° C. for 30 secondsand at 72° C. for 30 seconds was carried out 15 times using N5KG2 as atemplate and primers IgG2Bam: atatggatccTCATTTACCCGGAGACAGGGAGAGGCTC(SEQ ID NO: 6) and G2Ser1: GGCCTCCCAGCCTCCATCGAGAAAAC (SEQ ID NO: 7).The amplified DNA fragments were purified using a PCR purification kit,and the same amounts of the two purified DNA fragments were mixed.Thereafter, reaction at 98° C. for 1 second, at 60° C. for 30 secondsand at 72° C. for 30 seconds was carried out five times. Primers IgG3Nheand IgG2Bam were added to the mixture, and the same reaction was carriedout 15 times. The amplified DNA fragment was cleaved with NheI andBamHI, and substituted with the IgG1 constant region of the N5KG1 vector(N5KG2Ser). The fragment containing the sequence of the antibodyvariable region digested with BglII and NheI was incorporated into theN5KG2Ser vector.

The antibody expressed and purified by the above method was evaluated interms of binding ability to Ramos cells (FIG. 3A) and agonistic activity(FIG. 3B). The fluctuation in activity due to introduction of the P331Svariation was not observed.

Example 9 Measurement of CDC Activity of Anti-CD40 Agonistic Antibody331 Ser Mutant

CDC activity was measured by the above method. A rabbit serum-derivedcomplement was used, and Ramos cells were used as target cells. Theresults confirmed that, in a KM341-1-19 antibody at an antibodyconcentration of 1 μg/ml, IgG2ser exhibited CDC activity significantlyreduced as compared with IgG2 (FIG. 4A). On the other hand, when a humansupplement was used, no change was observed (FIG. 4B).

Example 10 Preparation and Activity Measurement of Agonist Anti-CD40Antibody Having Constant Region Converted

Among the anti-CD antibodies described in WO 02/088186, two antibodiesexhibiting strongest agonistic activity (KM341-1-19 antibody and 2105antibody) belong to IgG2 subclass. In order to examine whether or notthe IgG2 subclass is important for activation of CD40, recombinantproteins having an antibody constant region converted into IgG1, IgG3and IgG4, respectively, were prepared, and measured in terms of bindingability to an antigen and CD95 expression enhancing activity in Ramoscells according to Examples 4 and 6. IgG1 was expressed using N5KG1, andIgG2 and IgG3 were respectively expressed using expression vectors N5KG2and N5KG3 obtained by substituting the N5KG1 constant region with IgG2and IgG3, respectively. cDNA cloning of the IgG3 constant region wascarried out according to the IgG2 cloning method partially modified,using an IgG3-specific primer. IgG4 was expressed using N5KG4PE (IDECPharmaceuticals).

The antibody protein was expressed according to Example 1. Bindingactivity to Ramos cells expressing human CD40 of the KM341-1-19 antibodyand the 2105 antibody was not affected by converting IgG2 into IgG1,IgG3 or IgG4 (FIGS. 5A-1 and 5A-2). However, these antibodies were foundto have CD95 expression enhancing activity in Ramos cells reduced by 10%or more (FIGS. 5B-1 and 5B-2). This shows that not only the structure ofthe variable region defining the binding region of the antibody but alsothe structure of the constant region of the antibody are important forthe strong agonistic activity of the 2105 antibody and the KM341-1-19antibody. Thus, in order to examine which region in the IgG2 constantregion is important for agonistic activity, a domain swap mutant inwhich the IgG2 structure is mixed with the IgG4 structure was preparedto measure its activity. As described below, a domain swap mutant isprepared by substitution of a hinge region. In this case, the “hingeregion” includes the upper hinge (from Kabat EU code 216), middle hinge(from Kabat EU code 226) and lower hinge (Kabat EU code 231), asdescribed Ole H Brekke et. al., Immunology Today 1995, 16, 85-90. Fourdomain swap mutants IgG2/4 (CH1 and hinge region: IgG2, other regions:IgG4), IgG4/2/4 (hinge region: IgG2, other regions: IgG4), IgG2/4/4(CH1: IgG2, other regions: IgG4) and IgG4/2/2 (CH1: IgG4, other regions:IgG2) were prepared respectively for the KM341-1-19 antibody and the2105 antibody.

A vector N5KG2/4 for expressing the IgG2/4 antibody was prepared using aZtaq PCR kit (Takara). Reaction at 98° C. for 1 second, at 60° C. for 30seconds and at 72° C. for 30 seconds was carried out 15 times usingN5KG2 as a template and primers IgG3Bam:atatggatccTCATTTACCCGGAGACAGGGAGAGGC (SEQ ID NO: 8) and 24Chi4:AGGGGTCCGGGAGATCATGAGAGTGTCCTT (SEQ ID NO: 9). At the same time,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out 15 times using N5KG4 (IDECPharmaceuticals) as a template and primers 24Chi3:AAGGACACTCTCATGATCTCCCGGACCCCT (SEQ ID NO: 10) and linkH2:tgatcatacgtagatatcacggc (SEQ ID NO: 11). The amplified DNA fragmentswere purified using a PCR purification kit, and the same amounts of thetwo purified DNA fragments were mixed. Thereafter, reaction at 98° C.for 1 second, at 60° C. for 30 seconds and at 72° C. for 30 seconds wascarried out five times. Primers IgG3Bam and linkH2:tgatcatacgtagatatcacggc (SEQ ID NO: 12) were added to the mixture, andthe same reaction was carried out 15 times. The amplified DNA fragmentwas cleaved with NheI and BamHI, and substituted with the IgG1 constantregion of the N5KG1 vector.

A vector N5KG4/2/4 for expressing IgG4/2/4 was prepared as follows.Reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out 15 times using N5KG4 as a template andprimers linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 13), G2Hin3:TTTGCGCTCAACTGTCTTGTCCACCTTGGTGTTGCTGGG (SEQ ID NO: 14), linkH2:tgatcatacgtagatatcacggc (SEQ ID NO: 15) and G2Hin4:ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCG (SEQ ID NO: 16). The amplified DNAfragments were purified with a PCR purification kit, and the sameamounts of the two purified DNA fragments were mixed. Thereafter,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out five times using the mixture as atemplate. Primers linkH and linkH2 were added to the mixture, and thesame reaction was carried out 15 times. The amplified DNA fragment wascleaved with NheI and BamHI, and substituted with the IgG1 constantregion of the N5KG1 vector.

A vector N5KG2/4/4 for expressing IgG2/4/4 was prepared as follows.Reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out 15 times using N5KG2 as a template andprimers linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 17) and G4CH1-2:GGTGTTGCTGGGCTTGTGATCTACGTTGCAG (SEQ ID NO: 18). At the same time,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out 15 times using N5KG4 as a template andprimers G4CH1-1: CTGCAACGTAGATCACAAGCCCAGCAACACC (SEQ ID NO: 19) andlinkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 20). The amplified DNAfragments were purified using a PCR purification kit, and the sameamounts of the two purified DNA fragments were mixed. Thereafter,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out five times. Primers linkH and linkH2 wereadded to the mixture, and the same reaction was carried out 15 times.The amplified DNA fragment was cleaved with NheI and BamHI, andsubstituted with the IgG1 constant region of the N5KG1 vector.

A vector N5KG4/2/2 for expressing IgG4/2/2 was prepared as follows.Reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out using N5KG4 as a template and primerslinkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 21) and G4CH1-2:GGTGTTGCTGGGCTTGTGATCTACGTTGCAG (SEQ ID NO: 22). At the same time,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out 15 times using N5KG2 as a template andprimers G4CH1-1: CTGCAACGTAGATCACAAGCCCAGCAACACC (SEQ ID NO: 23) andlinkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 24). The amplified DNAfragments were purified using a PCR purification kit, and the sameamounts of the two purified DNA fragments were mixed. Thereafter,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out five times. Primers linkH and linkH2 wereadded to the mixture, and the same reaction was carried out 15 times.The amplified DNA fragment was cleaved with NheI and BamHI, andsubstituted with the IgG1 constant region of the N5KG1 vector.

Binding activity of the respective four domain swap mutants of theKM341-1-19 antibody and the 2105 antibody was examined. As a result, nodifference between them and the original IgG2 was observed in terms ofbinding ability (FIGS. 6A-1 and 6A-2). However, only IgG2/4/4 of boththe KM341-1-19 antibody and the 2105 antibody exhibited significantlyreduced agonistic activity (FIGS. 6B-1 and 6B-2). The results confirmedthat the hinge region of IgG2 is important for agonistic activity.

Further, it was examined which sequence is important in the hingeregion. The hinge region is divided into three sites, specifically,upper hinge, middle hinge and lower hinge (Ole H Brekke et al.Immunology Today 1995, 16, 85-90). IgG2-specific sequences in theseregions were respectively substituted with IgG4-specific sequences.Antibodies obtained by introducing a mutation into the upper hinge (fromKabat EU code 216), middle hinge (from Kabat EU code 226) and lowerhinge (from Kabat EU code 231) were respectively defined as IgG2UH4,IgG2MH4 and IgG2LH4. Their respective expression vectors were defined asN5KG2UH4, N5KG2 MH4 and N5KG2LH4. “Hinge” is defined as EU indices 216to 230 according to Kabat et al., Sequences of proteins of immunologicalinterest, 1991 Fifth edition.

N5KG2UH4 was prepared as follows. Reaction at 98° C. for 1 second, at60° C. for 30 seconds and at 72° C. for 30 seconds was carried out 15times using N5KG2 as a template and primers linkH:gggtacgtcctcacattcagtgatcag (SEQ ID NO: 25) and UH4-2:CACAACATTTggaCTCAACTcCTTGTCCACC (SEQ ID NO: 26). At the same time,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out 15 times using N5KG2 as a template andprimers UH4-1: GGTGGACAAGAgAGTTGAGtccAAATGTTGTG (SEQ ID NO: 27) andlinkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 28). The amplified DNAfragments were purified using a PCR purification kit, and the sameamounts of the two purified DNA fragments were mixed. Thereafter,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out five times. Primers linkH and linkH2 wereadded to the mixture, and the same reaction was carried out 15 times.The amplified DNA fragment was cleaved with NheI and BamHI, andsubstituted with the IgG1 constant region of the N5KG1 vector.

N5KG2 MH4 was prepared as follows. Reaction at 98° C. for 1 second, at60° C. for 30 seconds and at 72° C. for 30 seconds was carried out 15times using N5KG2 as a template and primers linkH:gggtacgtcctcacattcagtgatcag (SEQ ID NO: 29) and UM4-2:GGCACGGTGGGCAtgggggaccataTTTGCGCTC (SEQ ID NO: 30). At the same time,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out 15 times using N5KG2 as a template andprimers UM4-1: GAGCGCAAAtatggtcccccaTGCCCACCGTGCC (SEQ ID NO: 31) andlinkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 32). The amplified DNAfragments were purified using a PCR purification kit, and the sameamounts of the two purified DNA fragments were mixed. Thereafter,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out five times. Primers linkH and linkH2 wereadded to the mixture, and the same reaction was carried out 15 times.The amplified DNA fragment was cleaved with NheI and BamHI, andsubstituted with the IgG1 constant region of the N5KG1 vector.

N5KG2LH4 was prepared as follows. Reaction at 98° C. for 4 second, at60° C. for 30 seconds and at 72° C. for 30 seconds was carried out 15times using N5KG2 as a template and primers linkH:gggtacgtcctcacattcagtgatcag (SEQ ID NO: 33) and UL4-2:GAAGACTGACGGTCCccccaggaactcTGGTGCTGGGCA (SEQ ID NO: 34). At the sametime, reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at72° C. for 30 seconds was carried out 15 times using N5KG2 as a templateand primers UL4-1: TGCCCAGCACCAgagttcctggggGGACCGTCAGTCTTC (SEQ ID NO:35) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 36). The amplifiedDNA fragments were purified using a PCR purification kit, and the sameamounts of the two purified DNA fragments were mixed. Thereafter,reaction at 98° C. for 1 second, at 60° C. for 30 seconds and at 72° C.for 30 seconds was carried out five times. Primers linkH and linkH2 wereadded to the mixture, and the same reaction was carried out 15 times.The amplified DNA fragment was cleaved with NheI and BamHI, andsubstituted with the IgG1 constant region of the N5KG1 vector.

The three respective domain swap mutants of the KM341-1-19 antibody andthe 2105 antibody were examined to have the same binding activity to anantigen (FIGS. 6A-1 and 6A-2). However, IgG2UH4 and IgG2 MH4 exhibitedsignificantly reduced agonistic activity to Ramos cells (FIGS. 6B-1 and6B-2). It was found from the above that the structures of upper hingeand middle hinge in the hinge region are important for IgG2subclass-dependent agonistic activity of the anti-CD40 antibodiesKM341-1-19 and 2105.

Since IgG2 subclass was found to be important for agonistic activity,antibodies of subclass other than IgG2 were converted to those of IgG2subclass to examine whether or not the agonistic activity was enhanced.In the examination on several clones, agonistic activity of F76 could beenhanced by converting IgG1 subclass to IgG2 subclass (FIGS. 7A and B).

Example 11 Preparation of Anti-CD40 Antagonist Antibody Mutants

A DNA fragment containing a heavy chain and a light chain of a 4D11antibody gene described in WO 02/088186, whose original subclass isIgG1, was digested with BglII and NheI, purified, and then integratedinto N5KG4PE, N5KG4P and N5KG4 vectors (IDEC Pharmaceuticals). N5KG4PEcontains point mutations S228P and L235E in the IgG4 constant region,and N5KG4P contains a point mutation S228P in the IgG4 constant region.The antibody protein was expressed and purified according to the abovemethod. The antibody was purified according to the above method usingbinding to Ramos cells as an index. Change in binding activity of IgG1,IgG4, IgG4P and IgG4PE to Ramos cells was not observed (FIG. 8A).Antagonistic activity of IgG1 was compared with those of various IgG4mutants according to the above method to find that antagonistic activityof IgG1 does not differ from those of the IgG4 mutants (FIG. 8B).

Example 12 Evaluation of ADCC Activity and CDC Activity of Anti-CD40Antagonist Antibody Mutants

ADCC activity and CDC activity of anti-CD40 mutant antibodies wereevaluated according to the above method.

When using human MNC as effector cells and CD40-expressing Daudi cellsas target cells, two mutants IgG4 and IgG4PE were respectively observedto have ADCC activity significantly reduced as compared with IgG1 as theoriginal subclass of the 4D11 antibody (FIG. 9).

CDC activity of IgG1 was compared with that of IgG4P using Daudi cellsas target cells. IgG4P was found to have CDC activity significantlyreduced as compared with IgG1 (FIG. 10).

Example 13 Effect of Anti-CD40 Antagonistic Antibody on B Cells

100 μg each of IgG1, IgG4P and IgG4PE of the 4D11 antibody wasadministered to the tail vein of mice having a genetic backgroundwhereby they were homozygotes for mouse endogenous disrupted CD40 andharboring a transgene of a human CD40 gene (Yasui. et al. Int. Immunol.2002 Vol 14: 319). 24 hours after the administration, blood wascollected from the orbital venous plexus. After hemolysis with 0.16mol/L of ammonium chloride, an FITC-labeled anti-B220 antibody was addedto the hemolysate, and it was analyzed using FACS. The results are shownin FIG. 11. In the figure, the longitudinal axis indicates the ratio ofB cells in the total lymphocytes. IgG1 reduced the ratio of B cellsmost, IgG4P reduced the ratio to a lesser extent, and IgG4PE reduced theratio to a much lesser extent. 24 hours after the administration, thespleen was removed and crushed with a slide glass to prepare a cellsuspension. After hemolysis of the cell suspension, a PE-labeledanti-B220 antibody and an FITC-labeled anti-CD23, CD86 or CD95 antibodywere used for the hemolysate, and it was analyzed using FACS. Theresults are shown in FIGS. 12A, B and C. In the figures, thelongitudinal axis indicates the ratio of B cells expressing each surfacemarker in the total lymphocytes. 4D11G1 was found to achieve the samelevel of increase in expression of each marker as in a commerciallyavailable mouse anti-human CD40 agonistic antibody 5C3 (Pharmingen).IgG4PE achieved a smaller increase in expression of each activationsurface marker as compared with IgG1 and IgG4P.

Example 14 Effect of Inhibiting Production of Antigen-Specific Antibodyand Change in the Number of B Cells Caused by Anti-CD40 AntagonisticAntibody

100 μg (based on NP-CGG) of a complex of4-hydroxy-3-nitrophenylacetyl-chiken γ-globulin conjugates (NP-CGG:distributed by Professor Hitoshi KIKUTANI, Research Institute forMicrobial Diseases, Osaka University) and alum (aluminum hydroxide gel)was intraperitoneally administered to mice having a genetic backgroundwhereby they were homozygotes for mouse endogenous disrupted CD40 andharboring a transgene of a human CD40 gene (Yasui et al. Int. Immunol.2002 Vol 14: 319) to sensitize the mice. Immediately before the antigensensitization, 50 or 100 μg of each antibody was administered to thetail vein. 100 μg of an anti-human albumin human IgG4PE antibody wasadministered as a negative control. 7 and 14 days after thesensitization, blood was collected from the orbital venous plexus. Theamounts of NP-specific IgG1 and IgM antibodies in the serum weremeasured by the ELISA method. The ELISA method was carried out asfollows. 50 μl/well of NP-bound bovine serum albumin (NP-BSA: 2.5 μg/ml)was added to each well of a 96-well microplate for ELISA (Maxisorp,manufactured by Nunc A/S) and incubated at 4° C. to cause NP-BSA to beadsorbed thereon. Next, the supernatant was discarded, and a blockingreagent (SuperBlock, manufactured by Pierce Biotechnology, Inc.) wasadded to each well and incubated at room temperature to carry outblocking. Then, each well was washed with a phosphate buffer (PBS-T)containing 0.1% Tween 20 three times. Next, each serum diluted withPBS-T containing 10% Block Ace (50 μl/well) was added to each well, andincubated and reacted at 37° C. for two hours. The microplate was washedwith PBS-T three times. Then, a 1,000-fold dilution of a goat anti-mouseIgG1 antibody or IgM antibody labeled with alkaline phosphatase (CosmoBio, 1070-04 or 1020-04) with PBS-T containing 10% Block Ace (50μg/well) was added to each well, and incubated at 37° C. for two hours.Next, the microplate was washed with PBS-T, and then a coloringsubstrate solution (50 μl/well, Sigma 104, phosphatase substrate) wasadded to each well. The absorbance at a wavelength of 405 nm wasmeasured using a microplate reader. The results are shown in FIGS. 13Aand B. In the figures, the longitudinal axis indicates values obtainedby converting a 10,000-fold dilution (in the case of IgG1) or 100-folddilution (in the case of the IgM antibody) of serum collected fromC57BL/6 mice, to which NP-CGG was injected twice, and pooled into oneunit. The 4D11 antibody and the IgG4P or IgG4PE antibody of 281inhibited production of NP-specific IgG1 and IgM antibodies equallystrongly.

The change in the number of B cells in the peripheral blood and spleenin the mice used for examining the effect of inhibiting antibodyproduction was measured according to the same method as in Example 1.The results are shown in FIGS. 14A and B. The 4D11 antibody and theIgG4P antibody of 281 reduced the ratio of B cells in peripheral bloodsignificantly as compared with the IgG4PE antibody. Administration of100 μg of the IgG4PE antibody did not change the ratio of B cells in thespleen removed 14 days after the antigen sensitization. However,administration of IgG4P changed or tended to change the ratio.

Example 15 Effect of Anti-CD40 Antagonistic Antibody on CynomolgusMonkeys

30 mg/kg of IgG4P or IgG4PE of a 4D11 antibody was administered to theforearm cephalic vein of cynomolgus monkeys, and blood was collectedfrom the femoral vein after a certain period of time. In the subsetanalysis of peripheral blood lymphocytes, an FITC-labeled anti-CD3antibody, PE-labeled anti-CD20 antibody and APC-labeled anti-CD45antibody were used for each cell suspension, and the ratio of positivecells was measured using FACS to calculate the ratio of CD45 positivecells. The results are shown in FIG. 15. In the figure, the longitudinalaxis indicates the ratio of CD20 positive cells at each time to CD20positive cells before antibody administration. 1 to 7 days after theantibody administration, CD20 positive cells were reduced by about 40%in individuals to which the IgG4P antibody was administered. However, 4days after the administration, CD20 positive cells were reduced by onlyabout 20% in individuals to which the IgG4PE antibody was administered.

The IL12 concentration in serum was measured by the ELISA method. Bloodcollected from the femoral vein was allowed to stand at room temperaturefor 20 to 60 minutes, and then centrifuged at 3,000 rpm at roomtemperature for 15 minutes. The IL12 concentration in the resultingserum was measured using a monkey IL12 ELISA kit (BioSourceInternational Inc.). The results are shown in FIG. 16. No increase inIL12 production by the IgG4PE antibody was observed at any bloodcollection point. However, maximum IL12 production by the IgG4P antibodywas observed on the 4th day.

Example 16 Effect of Anti-CD40 Antagonistic Antibody on CynomolgusMonkey Delayed Hypersensitivity Model

Nine male cynomolgus monkeys were intradermally and intramuscularlysensitized with Tetanus toxoid (TTx) (10 Lf/ml; Denka Seiken Co., Ltd.)to induce delayed hypersensitivity to TTx. At the same time, 10 minutesbefore the start of sensitization, 0.1 and 10 mg/kg of a 4D11G4PEantibody was intravenously administered to each three animals threetimes (once a week) to examine the effect of 4D11G4PE on delayedhypersensitivity. Under anesthesia by intramuscular administration ofketamine, sensitization was carried out by intradermal administration ofTTx to the back (50×12 sites) and intramuscular administration of TTx tothe femur (0.6 mL/body), and challenge was carried out by intradermaladministration of TTx to the thorax (10 μL/site, 0 to 10 Lf/ml for eachthree sites) 21 days after the sensitization. 24 and 48 hours after theelicitation, skin reaction at the administration sites was observed andevaluated according to the Draize skin irritation score. The results ofdetermining the TTx concentration in each three sites were respectivelya mean value. The results are shown in FIG. 17. Administration of the4D11G4PE antibody apparently inhibited the delayed hypersensitivityreaction observed 24 and 48 hours after the administration.

The effect of TTx on TTx-specific IgG and IgM antibody titers wasexamined. Blood collected from the femoral vein over time was allowed tostand at room temperature for 20 to 60 minutes, and then centrifuged at3,000 rpm at room temperature for 15 minutes. The antibody titer in theresulting serum was measured using the ELISA method. The ELISA methodwas carried out as follows. 100 μl/well of TTx (0.5 Lf/ml) was added toeach well of a 96-well microplate for ELISA (Maxisorp, manufactured byNunc A/S) and incubated at 4° C. to cause TTx to be adsorbed thereon.Next, the supernatant was discarded, and a blocking reagent (phosphatebuffer containing 0.5% BSA) was added to each well and incubated at roomtemperature to carry out blocking. Then, each well was washed with aphosphate buffer (PBS-T) containing 0.05% Tween 20 three times. Next,each serum diluted with PBS-T containing 0.5% BSA (100 to 819,200-folddilution, dilution magnification: 2; 100 μl/well) was added to eachwell, and incubated and reacted at room temperature for two hours. Themicroplate was washed with PBS-T three times. Then, a 3,000-folddilution of a goat anti-monkey IgG antibody or IgM antibody labeled withperoxidase (Nordic Immunology) with PBS-T containing 0.5% BSA (100μg/well) was added to each well, and incubated at room temperature forone hour. Next, the microplate was washed with PBS-T, and then acoloring substrate solution (100 μl/well, o-phenylenediaminehydrochloride+aqueous hydrogen peroxide) was added to each well. Theabsorbance at a wavelength of 492 nm was measured using a microplatereader. The anti-TTx antibody titer was defined as a maximum dilutionmagnification to make the absorbance 0.1 and more. The antibody titerwas 0 when the absorbance did not reach 0.1 even at 100-fold dilution.The results are shown in FIGS. 18 and 19. Administration of 1 mg/kg of4D11G4PE suppressed the TTx-specific IgG and IgM antibody titers toabout 1/10. When 10 mg/kg of 4D11G4PE was administered, the antibodytiters were below the detection sensitivity at any blood collectionpoint.

Example 17 Effect of Anti-CD40 Antagonistic Antibody on PlateletThrombus Formation

Blood collected from a healthy human was divided into four aliquots(each 6 ml). Control human IgG4PE, control mouse IgG2a, human anti-humanCD40 IgG4PE (4D11) and mouse anti-human CD154 IgG2a (5C8) wererespectively added to the fractions so that each fraction had a bloodconcentration of 100 μg/ml. A flat perfusion chamber (GlycoTech Corp.)and a collagen-coated Petri dish were assembled according to theattached instruction. The blood treated with various antibodies wascaused to flow into the chamber at a rate that can apply a shear stressof 1,500/s to the blood for seven minutes. Thereafter, a 4%paraformaldehyde phosphate buffer was caused to flow into the chamber ata rate that can apply a shear stress of 1,500/s to the buffer for 10minutes. The platelet aggregate formed on the Petri dish was fixed,stained with a platelet-specific PE-labeled CD41a antibody, and observedwith a fluorescence microscope. The results are shown in FIGS. 20A andB. The blood treated with human anti-human CD40 IgG4PE (4D11) formed aplatelet aggregate on the collagen-coated Petri dish, as the bloodtreated with the control antibodies did. However, the blood treated withmouse anti-human CD154 IgG2a did not form a platelet aggregate.

Example 18 Evaluation of Stability of Anti-CD40 Antagonistic Antibody

Constant region-modified antibodies of the 4D11 antibody were comparedand examined in terms of stability. In the evaluation method, culturesupernatants obtained by respectively transiently expressing G4P, G4PE,G2Ser and G4/2/4 in HEK293 cells were charged with a Protein A column(Amersham Pharmacia Biotech), eluted with a 0.1 M citrate buffer (pH2.7), and then incubated at 37° C. for 1 minute and 10 minutes.Thereafter, they are neutralized with a 50 nM phosphate buffer (pH 7.0).The oligomer content in the resulting antibody solutions was measuredusing a gel filtration column (Tosoh Corp.). As a result, it was foundthat the oligomer content increases in proportion with the incubationtime, and G4/2/4 produces an oligomer easiest, G4PE second easiest,G2Ser third easiest, and G4P fourth easiest (FIG. 21).

Example 19 Effect of Inhibiting Skin Graft Rejection by Anti-CD40Antagonistic Antibody

A graft collected from the tail of DBA/2 mice was grafted into the sidedorsal thorax of C57BL/6 background mice having a genetic backgroundwhereby they were homozygotes for mouse endogenous disrupted CD40 andharboring a transgene of a human CD40 gene, and the graft was fixed witha plaster for seven days. 100 μg of a test substance 4D11G4PE or avehicle was administered to the tail vein 0, 2, 4, 7, 9, 11 and 14 daysafter the skin graft, respectively. To inhibit graft rejection by NKcells, 100 μg of an anti-asialo GML antibody was intraperitoneallyadministered to all mice 3 days before the operation and 1, 4 and 9 daysafter the operation. The results are shown in FIG. 22. The delay ingraft rejection was observed to be significant in the4D11G4PE-administered group as compared with the vehicle-administeredgroup.

Example 20 Analysis of CD40 Expression in Human Tumor Cell Lines

Expression of CD40 in a Burkitt's lymphoma cell line Ramos, bladdercancer cell line T24 (ATCC, HTB-4), pancreatic cancer cell line Hs 766T(ATCC, HTB-134) and Capan-2 (ATCC, HTB-80) was confirmed by FACSanalysis using 341G2Ser.

T24, Hs 766T and Capan-2 were digested with trypsin and harvested, andRamos was harvested as is. The cell lines were washed with PBS, and thenre-suspended in a staining buffer containing 1 μg/ml of 341G2Ser. Thestaining buffer was prepared by adding 0.05 mM EDTA, 0.05% sodium azideand 5% immobilized bovine serum to PBS. After incubation at 4° C. for 15minutes, the cells were washed with the staining buffer twice, andre-suspended in a 1:250 dilution of PE-bound goat anti-human IgG (γ)(Southern Biotechnology Associates, Inc) with the staining buffer. Afterincubation at 4° C. for 15 minutes, the cells were washed with thestaining buffer twice, and analyzed with FACSCalibur™ (manufactured byBD Biosciences). The same amount of a human anti-2,4-dinitrophenol (DNP)antibody was used as a negative control. The analysis was carried outusing Cellquest™ (manufactured by BD Biosciences) as data analysissoftware to calculate the mean fluorescence intensity.

As a result, Ramos, T24, Hs766T and Capan-2 had a mean fluorescenceintensity obviously higher than that of the negative control whenstained with 341G2Ser, and thus expression of CD40 was confirmed.

Example 21 Effect of Anti-CD40 Agonistic Antibody on Human Tumor CellLines

2.5×10³ Ramos cells, 2.5×10² T24 cells, 5×10³ Hs766T cells and 5×10³Capan-2 cells were respectively suspended in a medium to make the totalvolume of 100 μL in a flat bottom 96-well plate (manufactured byFalcon). Ramos and Hs766T, Capan-2 and T24 were cultured for 66 hours,90 hours and 114 hours, respectively, together with 341G2Ser at aconcentration of 1 ng/ml to 1,000 ng/ml at 37° C. in the presence of 5%CO₂. 10 μL (3.7 MBq/mL) of ³H-labeled thymidine (manufactured byAmersham Biosciences) was added and cultured at 37° C. in the presenceof 5% CO₂ for six hours. Ramos cells were harvested on Printed FiltermatA (manufactured by PerkinElmer, Inc.) using a 96 micro cell harvester(manufactured by Skatron Instruments, Inc.), and covered with a samplebag (manufactured by PerkinElmer, Inc.). 12 mL of Betaplate Scint(manufactured by PerkinElmer, Inc.) was added, and the β-ray dose wasmeasured with a liquid scintillation counter (Pharmacia 1205 Betaplate:manufactured by Pharmacia Corp.). Hs 766T cells, T24 cells and Capan-2cells were respectively harvested on Unifilter (manufactured byPerkinElmer, Inc.) using a harvester (manufactured by PerkinElmer,Inc.). A special seal was attached to the back of each filter, and 20μL/well of MicroScint 20 (manufactured by PerkinElmer, Inc.) was addedthereto. The β-ray dose was measured with a scintillation counter(TopCount: manufactured by Packard Instrument Co., Inc.). Data wereexpressed as cell survival rates (%) obtained by dividing a mean oftriplicate measurements obtained in three independent tests by a valueof non-treatment control.

As a result, the cell survival rates were reduced in all cell linesdepending on the 341G2Ser concentration (Table 1). When adding 100 ng/mlof 341G2Ser, the Ramos cell survival rate was 58%, the T24 cell survivalrate was 22%, the Hs 766T cell survival rate was 15%, and the Capan-2cell survival rate was 77%. 341G2Ser was found to have activity ofinhibiting growth of Ramos cells, T24 cells, Hs 766T cells and Capan-2cells.

TABLE 1 Cell survival rate 341G2Ser concentration (ng/ml) Cell line 1 10100 1000 Ramos 98.49% 81.68% 57.77% 55.26% T24 97.94% 50.72% 21.97%25.35% Hs 766T 34.67% 21.50% 14.67% 15.18% Capan-2 100.94% 85.34% 76.76%72.89%

Example 22 Effect of Anti-CD40 Agonistic Antibody on MouseCancer-Bearing Model (1) Ramos Cells

Six-week-old female Balb/c nude mice (purchased from CLEA Japan, Inc.)were irradiated with 3Gy radiation, and 2×10⁷ cells/mouse of Ramos cellswere subcutaneously grafted into the back thereof. 16 days after thegraft, the size of tumors that took there was measured. Cancer-bearingmice having a tumor size of 50 to 170 mm³ were classified into groupseach consisting of five mice. 100 μg/mouse of 341G2Ser (a solution in200 μl of PBS containing 1% nude mouse serum) was intravenouslyadministered to the cancer-bearing mice once on the 16th day, and thetumor size was measured until the 47th day. A human anti-human serumalbumin (HAS) antibody was used as a negative control.

(2) T24 Cells

A T24 cell mass that had undergone subcutaneous passage in the back ofnude mice three times were removed, and subcutaneously grafted into theback of six-week-old female Balb/c nude mice (purchased from CLEA Japan,Inc.). The tumor cell mass to be grafted is appropriately about 3 mmsquare. 10 days after the graft, the size of engrafted tumors wasmeasured. Cancer-bearing mice having a tumor size of 80 to 200 mm³ wereclassified into groups each consisting of five mice. 100 μg/mouse of341G2Ser (a solution in 200 μl of PBS containing 1% nude mouse serum)was intravenously administered to the cancer-bearing mice once on the10th day, and the tumor size was measured until the 29th day. The sameamount of a human anti-DNP antibody was used as a negative control.

(3) Hs 766T Cells

7×10⁵ cells/mouse of Hs 766T cells were subcutaneously grafted into theback of eight-week-old female Balb/c nude mice (purchased from CLEAJapan, Inc.). 16 days after the graft, the size of engrafted tumors wasmeasured. Cancer-bearing mice having a tumor size of 50 to 140 mm³ wereclassified into groups each consisting of five mice. 100 μg/mouse of341G2Ser (a solution in 200 μl of PBS containing 1% nude mouse serum)was intravenously administered to the cancer-bearing mice once on the16th day, and the tumor size was measured until the 32nd day. The sameamount of a human anti-DNP antibody was used as a negative control.

(4) Capan-2 Cells

2×10⁶ cells/mouse of Capan-2 cells were subcutaneously grafted into theback of six-week-old female Balb/c nude mice (purchased from CLEA Japan,Inc.). 13 days after the graft, the size of engrafted tumors wasmeasured. Cancer-bearing mice having a tumor size of 30 to 130 mm³ wereclassified into groups each consisting of five mice. 10 or 100 μg/mouseof 341G2Ser (a solution in 200 μl of PBS containing 1% nude mouse serum)was intravenously administered to the cancer-bearing mice twice a weekfrom the 13th day, and the tumor size was measured until the 34th day. Ahuman polyclonal antibody (hIgG) (manufactured by Sigma Co.) was used asa negative control.

The tumor growth inhibition ratio (TGIR) was calculated from thefollowing formula.

100−[{(mean tumor volume of 341G2Ser-administered group on the lastmeasurement day−mean tumor volume of 341G2Ser-administered group on theday of start of antibody administration)/(mean tumor volume of negativecontrol-administered group on the last measurement day−mean tumor volumeof negative control-administered group on the day of start of antibodyadministration)}×100]

As a result, TGIR exceeded 100% in the T24, Hs766T and Capan-2cancer-bearing mice, and a decrease in the tumor volume was observed inthe mice. On the other hand, TGIR was 73.4% in the Ramos cancer-bearingmice, and an increase in the tumor volume was considerably suppressed inthe mice (Table 2). FIGS. 23 to 26 respectively show the change in thetumor volume of cancer-bearing mice to which Ramos cells, T24 cells, Hs766T cells and Capan-2 cells were respectively engrafted.

TABLE 2 Tumor growth inhibition ratio Amount of 341G2Ser administeredCell line 10 μg/head 100 μg/head Ramos — 73.40% T24 — 109.05% Hs 766T —108.55% Capan-2 103.49% 119.20%

The inhibition ratio in each cell line is a value on the lastmeasurement day.

INDUSTRIAL APPLICABILITY

As shown in the Examples, the anti-CD40 antibody of the presentinvention having a constant region into which a mutation is introducedand an anti-CD40 antibody in which a part of the structure of thesubclass is substituted with that of another subclass have reduced ADCCactivity and CDC activity, while maintaining its activity. Accordingly,the antibody of the present invention has the reduced cytotoxicity toCD40-expressing cells when administered to a subject as a therapeuticantibody, and thus can be used with safety.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference in their entirety.

Sequence Listing Free Text SEQ ID NOS: 2 to 36: Synthetic DNAs

SEQ ID NOS: 49 to 130: Synthetic peptides

1. An isolated monoclonal antibody that binds human CD40 and consists oftwo heavy chains, each consisting of an amino acid sequence ranging fromQ at position 27 to K at position 474 of SEQ ID NO: 140, and two lightchains, each consisting of an amino acid sequence raging from A atposition 23 to C at position 235 of SEQ ID No:
 142. 2. A pharmaceuticalcomposition that comprises the monoclonal antibody according to claim 1as an active ingredient.
 3. A method of treating or preventingautoimmune disease that comprises administering the monoclonal antibodyaccording to claim 1 to a patient.